Sergi Roberto's 95th minute winner completed Barca's comeback from a 4-0 first leg deficit at the Camp Nou on Wednesday.
"We were forced to pay out a six-figure sum on Barcelona to qualify," said leading British bookmaker Ladbrokes.
Barcelona stun PSG with mother of all comebacks
"It's the biggest in-play payout since Liverpool's famous night (in winning the Champions League from 3-0 down) in Istanbul 12 years ago, with the industry forced to hand over millions overall."
Ladbrokes reported three gamblers kept faith with the Spanish giants at 100/1 when they needed three goals in a thrilling final two minutes plus stoppage time.
The incredible turnaround saw Barca's odds to win the Champions League from before kick-off slashed from 40/1 to 11/4 favourites ahead of Real Madrid and Bayern Munich.
This comeback will never be forgotten, says Barcelona's Enrique
Barca's destruction allied to another Champions League fightback for Borussia Dortmund against Benfica landed one fortunate punter a £117,587 payout from Ladbrokes as his 17-team accumulator came in.
However, William Hill were counting their blessings by the lack of action they took on Barca with just seven percent of bets they took backing the Catalans to make history.
One wise gambler could be in line for a £6,000 reward, though, should Barca go on to lift the trophy in Cardiff in June, though, having put £180 on Luis Enrique's men at 33/1.
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