The Dutch rider edged ahead at the end of a pulsating 140km race, which featured two climbs of Surrey's Box Hill.
With the peloton cast adrift, Great Britain's Lizzie Armitstead was in line for the Host Nation's first medal of the Games, but the question remained which colour.
Armitstead positioned herself behind Vos entering the finishing straight, but the Dutchwoman was strong enough to hold off the Briton, who had to settle for second.
Russia's Olga Zabelinskaya was third.
I knew I had a big chance but I also knew that if I made a little mistake then Lizzie would take the gold.Marianne Vos
Vos, who suffered a broken collarbone just two months ago, said after the dramatic sprint finish: 'I knew Lizzie was really fast on the line so I was not at all confident.
'I knew I had a big chance but I also knew that if I made a little mistake then Lizzie would take the gold.
'So I had to choose the right moment at the finish and, yeah, I think I did.'