The new record surpassed both the previous club mark of 60,739 fans set in 2019 at Atletico Madrid's Metropolitano stadium, where the hosts beat Barca 2-0 in a league match, and the official attendance record for a women's game.
That was set at the 1999 World Cup final between the United States and China, when 90,195 fans were in attendance at the Rose Bowl in Pasadena.
All 99,354 tickets for Wednesday's match had been sold out since January after a huge engagement campaign by the club.
Yet expectations that the record would be broken were shaken after heavy rain fell all day in Barcelona and the match started with the stadium only half full.
But as the game progressed, fans slowly arrived and ended up almost filling the iconic Camp Nou, Europe's largest stadium, creating a fantastic atmosphere.
There was an enormous mosaic in the stands with the slogan 'More than empowerment'.
Barcelona, who won the quarter-final first leg 3-1 in Madrid, took the lead eight minutes into the first half with a brilliant curling shot from long range by Mapi Leon but Real fought back to lead 2-1 with goals from Olga Carmona and Claudia Zornoza.
Yet Barca recovered early in the second half with Aitana Bonmati, Claudia Pina and Alexia Putellas putting the hosts back in front with three goals in 10 minutes. Caroline Hansen rounded off the scoring in what was an electrifying match.
"I'm speechless. It's a dream come true. I can only answer with more work, getting better and winning games so we can give them a big spectacle," said Putellas.
"I saw a lot of girls, children with that spark in their eyes. It's amazing to be part of history."
