Defending champion Bolt caused shock in the stadium in Daegu as he came out of his blocks well before the gun.
Blake clocked 9.92 seconds to lead home American Walter Dix (10.08) and 2003 world champion Kim Collins (10.09).
But much of the talk will centre on the fate of Bolt, who still has the 200m and 4x100m to come.
The triple world and Olympic champion beat a hasty retreat after his misfortune, although was quoted as saying: "Looking for tears? "Not going to happen. I'm OK."
Bolt, 25, later released a short comment via the IAAF website, saying: "I have nothing to say right now. I need some time."
Asked about the defence of his 200m title which begins on Friday, Bolt added: "How will I go? We'll have to see on Friday."
Bolt was his usual relaxed self before the race, preening when his name was announced, and the outcome will no doubt lead people to question both his preparation and the false-start rule.
While Blake, 21, had sympathy for his training partner he nevertheless was overwhelmed by his unexpected triumph.
"I don't think I can find words to explain it, I feel like I want to cry," he said. "I have been praying for this moment, this is all a dream for me right now.
Previously, athletes had been allowed one false start before being disqualified for a second, but new rules were introduced by governing body the IAAF for the start of the 2010 season.
The 100m in Daegu had already been deprived of the three previous fastest men this year - Bolt's team-mates Asafa Powell and Steve Mullings and American Tyson Gay.
Britain's Dwain Chambers was disqualified for a false-start in the semi-finals, one day after compatriot Christine Ohuruogu was eliminated from the 400m for the same infringement.