Officials said the attack took place in Ratchaburi province on a road 100 kilometres (62 miles) west of the capital, Bangkok.
Three men opened fire with M-16 rifles on the bus, which was carrying more than 20 children from the Ban Kha Witthaya secondary school, just after 0700 on Tuesday (midnight GMT).
The driver of the bus managed to swerve away from the gunmen, who stepped out in front of it and sprayed it with bullets.
"I heard a series of loud shots and many of my friends were hit," a 13-year-old girl told a radio station.
"My school uniform was splattered with their blood."
Border closed
The gunmen were said to be wearing masks and jungle fatigues as worn by ethnic Karen guerrillas who operate in the area.
However the main Karen militia, the Karen National Union (KNU) has denied any involvement.
"It was a foolish act and the KNU has never done anything like this," KNU Secretary-General Pado Manh Sha told the AFP news agency.
A high-ranking member of the Vigorous Burmese Student Warriors, an extremist dissident group fighting for independence from Burma, also denied involvement.
"We deeply regret the deplorable incident. Our group denounces the attack," the member told AFP on condition of anonymity.
"I can guarantee that neither the student group, nor any members of the God's Army were involved in the attack," he said, referring to another extremist Karen group.
The BBC's Jonathan Head says there was no obvious motive for the killing.
The border has been closed for the last week due to a series of clashes between Thai and Burmese troops.
Ratchaburi province was the scene of a two-day hospital stand-off in January 2000 between government forces and rebels, in which all 10 rebels died.
Three months earlier, a group of Karen guerrillas were allowed to go free after taking hostages at the Burmese embassy in Bangkok.
The Karens have been fighting for independence from Burma since the country gained independence from Britain in 1948.