Vos clocked 3 hours, 44 minutes, 0 seconds along the 140-kilometer (87-mile) race route from Montecatini Terme to Florence.
Emma Johansson of Sweden finished second, 15 seconds behind, and Rossella Ratto of Italy crossed third with the same time.
Johansson also took the silver medal in the road race at the 2008 Beijing Olympics
While the Dutch and Italians had multiple riders in the lead pack at the end, Johansson had to fend for herself.
"I was quite isolated," she said. "My brain was working just as hard as my legs today. I tried to make all the right decisions. It's been a very hard race."
The 19-year-old Ratto gave host Italy its first medal of the championships.
"Since the start of the day I heard the fans cheering for me and then I saw a lot more people pushing for me when we got into Florence," Ratto said. "It's going to take a while for this to sink in."
The course concluded with five laps of a 16.6-kilometer (10.3-mile) hilly circuit in the Tuscan capital. Vos' attack came on the steepest section of the course, with a gradient of 16 percent. She then went into time trial mode for the final few kilometers (miles) and had enough time to lift her arms and celebrate even before she crossed the finish line.
"Until the last lap I didn't feel too good," Vos said. "On the long climb I wasn't the best but on the steep climb I knew I had a chance."
Only two women have won more titles in this race. French great Jeannie Longo-Ciprelli won it a record five times between 1985 and 1995 and Belgian standout Yvonne Reynders took four victories from 1959 to 1966.
Vos was also runner-up five straight times from 2007-11.
Vos' Dutch teammate Anna van der Breggen finished fourth, 33 seconds behind, and Evelyn Stevens of the United States was fifth, 46 seconds back.
Only 46 of the 141 starters finished.
"Italy's team was really strong today. And America, too," Vos said. "(The Americans) made it tough from the beginning when we arrived at the circuit. That's when we realized, 'Man, it's going to be a tough race.'
"It was great to have Anna there to react to some of the attacks," Vos added.
There was also a Dutch winner in the junior men's race, with Mathieu van der Poel covering the same course in 3 hours, 33 minutes, 14 seconds.
Van der Poel also attacked on the final lap of the circuit and had enough time to grab a Dutch flag and celebrate with it before he crossed the line.
Mads Pedersen of Denmark finished second, three seconds behind, and Iltjan Nika was third for Albania's first medal in any discipline, with the same time.
Van der Poel is the junior Dutch national road champion and a dual junior cyclo-cross world champion. His father, Adri van der Poel, was also an accomplished rider who won several single-day classics in the 1980s.
The worlds conclude Sunday with the featured men's pro race.
___
Follow Andrew Dampf at http://twitter.com/AndrewDampfAP
