The 20-year-old Team UAE rider is stronger climber who will defend his lead on Saturday's 'Queen' stage over five Alpine climbs on the way to the ski resort of Thyon at an altitude of 2,076m.
Ayuso completed the flat 18.75km time trial in 25min 15sec in a highly rural setting in the French-speaking part of Switzerland, with less cornering than an urban route, and less road furniture.
American Matteo Jorgenson of Movistar was second at five seconds while Ayuso's British teammate Adam Yates was third at 17sec.
Ayuso leads the overall standings by 18sec form Jorgenson with world time-trial champion Tobias Foss, a Norwegian who rides for Jumbo-Visma, in third at 19sec.
Foss and prologue winner Josef Cerny set the early pace with Foss ending 25sec down in eighth.
After Saturday's likely decisive stage, Sunday's finale is a 170km run to Geneva over two categorised climbs.
