The race, the first in this year's UCI Women's WorldTour and now regarded as a classic, covered 136km in and around Siena.
Van Vleuten crossed the finish line in the historic square ahead of Annika Langvad (Boels Dolmans) and Katarzyna Niewiadoma (Canyon-SRAM).
The victory was only the Dutchwoman’s second race after a serious knee injury suffered during the 2018 UCI Road World Championships.
“This is one of the best victories in my career. I thought after breaking my knee at the world championship that I wouldn’t have enough time to get into the right form for winning Strade Bianche,” Van Vleuten said.
“But I’m glad it was enough time. I managed to attack exactly where I had planned to.”
Van Vleuten made her move from the final selection of 11 riders on the steep climb, as the off-road sectors ended and the run to the finish began.
She quickly opened up a gap of 30 seconds and with no organised chase behind, the gap grew to 40 seconds as the climb to Piazza del Campo came into sight.
"I just waited for a good moment (to attack), there was only one teammate with me, only Lucy Kennedy, and it was really good for us to be there together but we were a bit outnumbered," Van Vleuten said.
"We were only two in a big group so we had to poker a bit, then I thought I had to put the hammer down on the uphill.”