The crossing to the Belgian port of Zeebrugge takes more than 17 hours, but will save passengers and truckers having to drive via Hull or the English Channel ports.
Chancellor Gordon Brown and Scotland's First Minister Jack McConnell took part in an inauguration ceremony on board the Superfast IX Ferry at the port on the Firth of Forth.
The boat was scheduled to leave the port at 1700BST, at the same time as another ferry departed from Belgium, but it left half an hour late to cheers from onlookers at the quayside.
Mr Brown, whose Dunfermline East constituency contains parts of Rosyth, described the occasion as a dream turned into reality.
He said he felt "extremely privileged" to mark the launch.
"Rosyth and Fife will now become the number one location for hundreds of exporters, hundreds of companies, increasing their trade links with the rest of Europe."
Each ferry can carry more than 1,000 passengers, 120 cars and 100 commercial vehicles.
Mr McConnell said the ferry route should remove up to two million lorry miles per year from Scottish roads by transferring trade to the seas.
He said: "I therefore encourage Scottish freight operators to make best use of the Rosyth service and make sure it's a success in the years to come."
The First Minister said the development would provide a major boost for the local economy and attract new businesses to locate in the area whilst also bringing Scotland closer to Europe.
Mr McConnell addressed fears that the new port could become an entry point for drugs into Scotland.
He said: "I think the appropriate authorities in the UK will be well aware of that concern and will make sure that this is a safe and secure location."
Great advantages
Phil Flanders of the Road Haulage Association said: "There is a lot of potential for people using the service.
"There is new drivers' hours regulations coming into effect in the next two years in the working times directives that is going to seriously restrict what hauliers can do.
"So this ferry gives them great advantages from Scotland and I just hope they give it a chance.
"We will be encouraging them to at least try the service out."