Amandine Henry scored a dazzling curled effort in the sixth minute, before Ada Hegerberg headed home an exquisite Selma Bacha cross to double Lyon's lead.
Catarina Macario tapped home a third from close range before Alexia Putellas pulled one back for the Spanish giants.
Despite pressure from Barca in the second half, Lyon remained resolute, once again being crowned European champions.
It was the battle of the two biggest teams in women's football.
Lyon: The seven-time winners packed full of experienced winners and led by prolific goal scorer Hegerberg.
And Barcelona: The most in-form team at the moment, which includes reigning Ballon d'Or Feminin winner Putellas.
The two sides have met before in a Champions League final, with Hegerberg scoring a hat trick in a 4-1 Lyon victory in 2019. But this time it was different.
And with a packed Juventus Stadium in Turin waiting, the hoards of fans were not disappointed.
Starting from the first minute, the match was an end-to-end affair, with both team's attacking prowess dominating proceedings.
But it was a little bit of magic -- fit for a final -- which had the fans on their feet early on.
Henry, picking up the ball almost 30 yards out, hit a stunning long-range shot which cannoned into the top corner, giving Barca goalkeeper Sandra Paños no chance.
That moment of class from France international Henry opened the floodgate for attacking forays, something the experienced heads of Lyon capitalized on.
A sumptuous left-footed cross from Bacha was nodded home by Hegerberg -- the competition's all-time top scorer -- to further extend the lead.
And the game looked to be out of sight for the reigning champions Barca when Macario tapped home from just inches out.
But, Barca -- a team that had made history just weeks ago when it completed a perfect league campaign comprised of 30 wins in 30 matches -- displayed its skill and grit to gain a foothold.
Putellas' goal late in the first half gave her side hope going into the halftime break.
After the break, it was more of the same, with chances for both sides, the closest coming when Patricia Guijarro attempted an audacious chip from almost 40 yards out, her effort slamming against the bar before bouncing away to safety.
Despite pressure from Barcelona, it was unable to finish any of its chances, meaning Lyon lifted its record-extending eighth Champions League title.