Kopecky, 26, scored his second win of the season.
At Strade Bianche, at the beginning of March, she had already beaten Van Vleuten.
The Dutch Chantal Van den Broek-Blaak, who caused the decision by attacking 11 kilometers from the finish, took third place in this 19th women's edition of the "Ronde".
"I was pretty confident for the sprint but you're never sure," said Kopecky.
"I told myself that I had done the hardest part by crossing the (11) mountains" of this 158 kilometer race.
The Belgian champion, who had placed third in 2020, succeeded Van Vleuten in the winners of the race.
She is the second Belgian to put her name on it after Grace Verbeke in 2010.
Ranking:
1. Lotte Kopecky (BEL / SD Worx), the 158 km in 4 h 11:21.
2. Annemiek van Vleuten (NED/Movistar) mt
3. Chantal Van den Broek-Black (NED/SD Worx) at 02.
4. Arlenis Sierra (CUB/Movistar) 40.
5. Marlen Reusser (SUI/SD Worx) 40.
6. Cecilie Ludwig (DEN/FDJ Nouvelle-Aquitaine) 40.
7. Grace Brown (AUS/FDJ New Aquitaine) 40.
8. Katarzyna Niewiadoma (POL/Canyon) 40.
9. Brodie Chapman (AUS/FDJ New Aquitaine) 42.
10. Marta Bastianelli (ITA/UAE) 1:10.
...
29. Aude Biannic (FRA/Movistar) 1:15.
© 2022 AFP