Pooley powered through the 22.9-km course in the port city of Geelong in 32 minutes 48.44 seconds to finish 15.17 seconds faster than German silver medallist Judith Arndt. Linda Villumsen of New Zealand took bronze.
Pooley, who took silver in the time trial at the 2008 Beijing Olympics, had to overcome a last-minute hitch when race officials found a discrepancy with her bike just before the starting line and ordered her to change it.
French cycling great Jeannie Longo-Ciprelli defied her 51 years with an extraordinary ride to finish fifth, ahead of a field of riders half her age.
Pooley will bid for the double in the road race on Saturday.
Taylor Phinney of the United States earlier dashed home-town hopes of a men's under-23 world champion in the time trial when he pipped Australia's Luke Durbridge by 1.9 seconds over the 31.6 km course.
Durbridge's silver at 19 years and 173 days made him the youngest under-23 world championship medalist, displacing Swiss three-times time trial world champion Fabian Cancellara, who was 33 days older when he won silver at Plouay in France, 10 years ago.
German rider Marcel Kittel captured the bronze.
