The 23-year-old Dutch rider, who had earlier won gold in the team time trial on Sunday, was last to set off across the 25.3km course from Le Cascine to Florence and by the first checkpoint had built up a lead of 20 seconds.
Van Dijk comfortably maintained her advantage to cross the finish line in first place to a great ovation from the crowd.
"I'm super happy. It's difficult to describe how I feel now. I'm so excited because I dreamt so long of this one and the pressure was high to finish it off. It's great to have won," she told Cycling News.
"My intention was to start fast but I wanted to keep going a bit longer than I did. I maybe got over excited and went too fast, but I maintained the time difference and so it was all ok."
New Zealand’s Linda Villumsen was 24 seconds back in silver with America’s Carmen Small edging out team-mate Evelyn Stevens for bronze by just four hundredths of a second.
1. Ellen van Dijk (Netherlands) 27:48
2. Linda Villumsen (New Zealand) +24
3. Carmen Small (USA) +28
4. Evelyn Stevens (USA) +28
5. Trixi Worrack (Germany) +31