The American, who finished behind Dutchwoman Leontien Zijlaard and ahead of Longo-Ciprelli in Sydney in the Olympic time trial, completed the 25-kilometer (15.6-mile) course in 33 minutes and 14 seconds.
"It's an achievement to become the world champion here," Holden said later. "It's incredible to beat Jeannie Longo two times."
Rasa Polikeviciute of Lithuania was third, 46 seconds off the lead pace.
Holden, a five-time U.S. champion in the time trial, held the lead throughout, and had a 16-second lead at the 5-kilometer mark.
"I didn't have to come here," Holden said. "I was very concentrated on the objective of a medal in Sydney. After the Olympic Games, I was very tired."
Ironically, it was Longo herself who persuaded Holden to participate in the championships in this town in Brittany, in western France. The two have been friends for five years. Longo often visits Colorado, home of the USA Cycling and Holden.
Dutchwoman Leontien van Moorsel, who won the time trial in Sydney, did not compete here.
In junior men time trials Wednesday, Poland's Peter Mazur won in 30 minutes, 58 seconds, with Russia's Vladimir Goussev placing second, 18 seconds behind. Poland's Lukasz Bodnar was third, 30 seconds behind.