Striker Samuel Eto'o, who left Barcelona for Inter Milan during the close-season, was fifth.
Messi played a key role as Barca won the treble of Champions League, domestic league and cup last season.
The 22-year-old said he was "really happy" to be the first Argentine winner of the award, but added: "I have to say that if the existing rules were in place years ago, Maradona would have won it."
The prize was only open to European footballers until 1995, when the rules were changed to include players from the rest of the world.
Argentina-born Alfredo Di Stefano won in 1957 and 1959 while at Real Madrid but he had acquired Spanish citizenship, Argentine-born Omar Sivori took the prize in 1961 after acquiring Italian citizenship.
Messi paid a warm tribute to Pep Guardiola, who led Barca to the treble in his first season as coach.
"I've always said that he's been a very important person, not only for me but for the whole group. There's a special relationship that is more than just a player-coach one," he said.
"The titles we won with him have been the key to winning the Ballon d'Or."
Argentina star Messi is in the running for Fifa World Player of the Year
Guardiola said what separated Messi from his rivals was his "competitive spirit".
"He has all the resources at his disposal: he's tough, quick, a goal-scorer, intelligent and reads the game well," he said.
"It is a prize for him and the team. He's a deserved winner and a player on a different level."
The highest-placed English player was Rooney, while Chelsea forward Didier Drogba came ninth.
France Football said Messi was only seven points short of the maximum available from the 96 jurors.
The magazine quoted Messi as saying: "Honestly, I knew that I was among the favourites because Barcelona had a fruitful year in 2009.
"But I didn't expect to win with such a margin.
"I dedicate it to my family. They were always present when I needed them and sometimes felt even stronger emotions than me."
Messi becomes the sixth Barcelona player to take the award but the first since Brazil playmaker Ronaldinho in 2005.
He recently signed a two-year contract extension with the European champions until 2016 - an improved deal which includes a buy-out clause set at 250m euros (£228m).
Messi top-scored in last season's Champions League with nine goals - one of them coming in his side's 2-0 victory over Manchester United in the final in Rome.
That United team contained Ronaldo, the 2008 Ballon d'Or winner, but the Portugal forward, who now plays for Real Madrid, was eclipsed by Messi on the night.
Barca coach Pep Guardiola, having completed an unprecedented treble of La Liga, Copa del Rey and Champions League titles, said after the final: "I think he is the best player and we didn't need to win today to know he is the best."
Messi, who joined the Catalan giants from Newell's Old Boys at the age of 13 in 2000, usually plays on the right flank even though he is left-footed.
His scampering runs off the wing and surges across the area to shoot or play-in a team mate have become a trademark of Barcelona's eye-catching attacking style.
1987: Born in Rosario, Santa Fe on 24 June
2000: Joins Barcelona as a 13-year-old from Newell's Old Boys youth team
2003: Makes senior debut at 16 in a friendly against Porto
2005: Leads Argentina to World Youth Championship title, collecting best player and top scorer trophies
2005: Makes Argentina debut in friendly against Hungary in August. Sent off for elbowing.
2005: Becomes a Spanish citizen in September
2005: Makes competitive debut for Barcelona in October
2005-06: Scores six goals in 17 appearances as Barca win Spanish league
2008: Wins gold medal with Argentina at Olympics
2008: Finishes second in the Fifa World Player of the Year vote
2009: Scores second goal in Champions League final victory over Manchester United in May as Barca win their third European Cup
2009: Signs a two-year contract extension in September to keep him at Barca until 2016
The arrival of right-back Daniel Alves from Seville in 2008 freed Messi from his wing duties, allowing him to move deeper into midfield, lose his markers, link up better with Barca playmakers Xavi and Iniesta, and burst through on goal from central positions.
He finished as the club's top scorer with 38 goals in all competitions last season.
Messi, whose style is usually likened to that of Argentine football legend Maradona, came through the Barca youth system and made his senior debut under Frank Rijkaard aged 16.
He is still only 22 but has amassed three La Liga titles, two European Cups and a Spanish cup. He helped Argentina win the Under-20 World Cup in 2005, finishing as top scorer, and won gold with his country at the 2008 Olympics.
Four years ago, Maradona, who has since become Argentina manager, said Messi would be the player to take over his mantle as national hero.
In addition to the Ballon d'Or, Messi is favourite to be crowned 2009 Fifa World Player of the Year on 21 December.
Final Ballon d'Or standings:
1. Lionel Messi (Argentina, Barcelona) 473 points 2. Cristiano Ronaldo (Portugal, Man United & Real Madrid) 233 3. Xavi (Spain, Barcelona) 170 4. Andres Iniesta (Spain, Barcelona) 149 5. Samuel Eto'o (Cameroon, Barcelona & Inter Milan) 75 6. Kaka (Brazil, AC Milan & Real Madrid) 58 7. Zlatan Ibrahimovic (Sweden, Inter Milan & Barcelona) 50 8. Wayne Rooney (England, Manchester United) 35 9. Didier Drogba (Ivory Coast, Chelsea) 33 10. Steven Gerrard (England, Liverpool) 32 11. Fernando Torres (Spain, Liverpool) 22 12. Cesc Fabregas (Spain, Arsenal) 13 13. Edin Dzeko (Bosnia, VfL Wolfsburg) 12 14. Ryan Giggs (Wales, Manchester United) 11 15. Thierry Henry (France, Barcelona) 9 16 Luis Fabiano (Brazil, Sevilla) 8 16 Nemanja Vidic (Serbia, Manchester United) 8 16 Iker Casillas (Spain, Real Madrid) 8 19. Diego Forlan (Uruguay, Atletico Madrid) 7 20 Yoann Gourcuff (France, Bordeaux) 6 21 Andrei Arshavin (Russia, Arsenal) 5 21 Julio Cesar (Brazil, Inter Milan) 5 21 Frank Lampard (England, Chelsea) 5 24 Maicon (Brazil, Inter) 4 25 Diego (Brazil, Werder Bremen & Juventus) 3 26 David Villa (Spain, Valencia) 2 26 John Terry (England, Chelsea) 2 28 Franck Ribery (France, Bayern Munich) 1 28 Yaya Toure (Ivory Coast, Barcelona) 1 30 Karim Benzema (France, Lyon & Real Madrid) 0
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