The American stopped the clock at 36 minutes and 21 seconds and moves into second overall behind Guarnier, who posted the fourth fastest time.
Stevens' time was just four seconds faster than Anna Van der Breggen (Rabobank Liv) and Elisa Longo Borghini (Wiggle High5).
"Coming into this, I really wanted to win the time trial. I don’t know if I’ve ever actually won a time trial in a stage race. I don’t think I have," Stevens said.
"To actually win it especially coming up to Rio and showing I deserve my spot and I’m ready to go – it’s awesome.”
“I’ve been climbing well so I decided I wanted to go as hard as I could to the top of the climb. I had Smiley [Boels-Dolmans soigneur Maciej Kowlewski] in my ear ‘You’re great. You’re great. But go a little faster, yeah?’," Stevens said.
American team-mate Guarnier said her focus for the stage was a strong effort to keep pink.
“I approached today’s effort in the same way as I did last year,” Guarnier said. “Although I lost the pink jersey in the time trial last year, I did a great personal effort, and that’s all you can do at the end of the day.”
When asked what went differently to last year, Guarnier said: “I kept pink!”
Stevens said she had no GC ambitions prior to the race.
“It’s funny. To be 1-2 is really nice. Karol-Ann [Canuel] and Amalie [Dideriksen] have done so much work already this week, and they’re going to have their hands full these next couple days but we’re in a good place," she said.