Longo, 52, beat Christel Ferrier Bruneau and Audrey Cordon - both young enough to be her daughters - on a 19km course in Boulogne-sur-Mer, northern France.
Longo, who has 14 Olympic or World gold medals on the road and track under her belt, won her first world title in 1985.
Europcar's Christophe Kern claimed the men's title.
Kern's time of 59:41 was a storming one, leaving Christophe Riblon (AG2R) 75 seconds adrift in second place.
Geoffroy Lequatre (Radioshack) settled for third place, while defending champion Nicolas Vogondy of Cofidis ended up 2:45 behind Kern's mark to take 10th place.
The French national road race championship event takes place on Sunday over 234.5km.
Elsewhere, Saxo Bank-SunGard's Gustav Larsson took his fourth Swedish national time trial championship as he thundered through the course one minute and 42 seconds ahead of second place Thomas Löfkvist (Sky).
High Road's Gatis Smukulis reigned supreme at the Latvian ITT Championships and BMC Racing Team's Martin Kohler won the gold at the Swiss national time trial championships.