Eugénie Le Sommer, Saki Kumagai and Sara Bjork Gunnarsdóttir scored for Lyon as it clinched a record-extending seventh Champions League trophy.
Wolfsburg, seeking its third title after back-to-back triumphs in 2013 and 2014, got on the board with Alex Popp after its French rival had opened a two-goal lead in the first half.
Wolfsburg also lost the final to Lyon in 2016 and 2018.
Le Sommer opened the scoring in the 25th minute before Kumagai added to the lead shortly before halftime.
Gunnarsdóttir sealed the victory in the 88th after Popp had got Wolfsburg on the board in the 58th.
“We played a great season, we can be proud, and I can proud to be part of this team,” Popp said.
Lyon's Le Sommer, Wendie Renard and Sarah Bouhaddi each won their seventh European title, moving them one clear of the men's record held by Paco Gento.
“It’s actually quite difficult to believe we have managed our fifth straight title, to be honest," Bouhaddi said. “We’re super proud of ourselves, it was a very difficult competition.”
It was the fourth final between the two clubs, and their third in the last five years. Wolfsburg’s only triumph over Lyon came in the first final between the teams in 2013.
It was Wolfsburg's first loss in 41 games, since a 4-2 home defeat in the Champions League quarterfinals against Lyon in March 2019. Lyon still hasn’t lost a competitive match since the French Cup final against Paris Saint-Germain in May 2018.
In the semifinals of the final-eight mini tournament played without fans in northern Spain, Lyon beat PSG and Wolfsburg defeated Barcelona.
The final was played at Real Sociedad’s Anoeta Stadium in the Basque city of San Sebastián. Athletic Bilbao’s San Mamés stadium also hosted games.