World Champion Italy's Giorgia Bronzini, center, celebrates as she crosses the finish line
The Italian finished ahead of Marianne Vos of the Netherlands and Germany's Ina-Yoko Teutenberg.
"I have no words," an exhausted Bronzini said after the race.
Canada's Clara Hughes earlier broke free of the peloton but was caught in the final kilometers.
The peloton had stuck together for most of the race. After 101 kilometers (63 miles), Namoi Cantele of Italy unsuccessfully attempted a breakout and the field ended more strung out in the hilly landscape near Holte, 19 miles (30 kilometers) north of Copenhagen.
After U.S. rider Amber Neben had an attempt in the eighth lap, Hughes, a former Olympic speedskater turned cyclist, took off and at one point was 46 seconds ahead. New Zealand's Linda Villumsen tried a pursuit but failed before a tired Hughes was caught in the last miles (kilometers).
Vos then spearheaded a Dutch attack followed by German and Italian riders, resulting in a mass sprint.
Dutch rider Vos now has five straight silver medals.
Villumsen, who grabbed silver in Tuesday's time trial, crashed with several riders in the last kilometers, including Denmark's Dorte Lohse.
In the junior world championships road race earlier Saturday, France's Pierre-Henri Lecuisinier won the sprint ahead of Belgium's Martijn Degreve and Steven Lammertink of the Netherlands.
Short link: