Sonia Bompastor's side came flying out of the blocks at the Juventus Stadium and were 3-1 up at half time courtesy of Amandine Henry, Ada Hegerberg and Catarina Macario's efforts.
2021 Ballon d'Or winner Alexis Putellas pulled one back for Barcelona near the end of the first half, and Jonatan Giraldez's side stepped up their game in the second period, but Lyon's defending was exemplary as they saw out a deserved win.
With only six minutes on the clock, Henry won the ball back a good 30 yards away from Sandra Panos's goal before unleashing a thunderbolt of an effort into the top corner.
Lyon then lost Ellie Carpenter to a knee injury, but the unfortunate blow did not halt their momentum by any means, as Hegerberg got on the end of Selma Bacha's sublime cross to head home and double her side's lead in the 23rd minute.
Barcelona were at sixes and sevens at the back and paid the price again just after the half-hour mark, as Hegerberg found Macario in acres of space at the back post for the American to tap home off the woodwork.
Putellas's clinical first-time finish gave Barcelona a route back into the game in the 41st minute, and the 2021 champions did improve after the break - with Patricia Guijarro very nearly scoring one of the best goals the Women's Champions League has ever seen.
The 24-year-old attempted to catch Christiane Endler out with a speculative effort from inside the centre circle, and she witnessed her wonderful effort crash back out off the crossbar.
Lieke Martens and Asisat Oshoala were both introduced as Barcelona desperately tried to claw their way back into the game in the dying embers, but it was Lyon who went closest as Hegerberg crashed a volley against the post with seconds remaining.
Lyon survived seven minutes of second-half injury time to dethrone the Catalans and steal back their European crown, making Bompastor the first woman to win the Champions League as a player and manager.