Making their sixth Champions League final appearance, Wolfsburg got off to the perfect start against the Catalan giants.
The competition's leading scorer Ewa Pajor scored her ninth Champions League goal of the season to give the German side a third-minute lead.
Wolfsburg looked to be heading towards a comfortable victory when Alexandra Popp nodded home from Pajor's exquisite cross.
However, Patri Guijarro clawed her side back from the brink of defeat, scoring two goals in as many minutes in the opening stages of the second half.
The turnaround was completed in the 70th minute when Fridolina Rolfo benefitted from a poor piece of defending to score the winning goal.
While Barcelona are crowned European champions for the second time, Wolfsburg have now lost their last four Champions League finals.
After conceding in the sixth minute of last season's Champions League final defeat to Lyon, Barcelona would have been desperate to avoid another poor start.
As it proved, the Spanish champions were caught napping once again, as Wolfsburg took a precious lead in the third minute.
Having recovered from a knee injury to make her first appearance since April, Lucy Bronze was not given the time to ease herself into the contest by a determined Pajor, who dispossessed the England international, before producing a thunderous effort into the top corner.
After getting off to a disappointing start, Barcelona went agonisingly close to finding an equaliser in the 13th minute.
Mapi Leon's deep corner kick found the head of her central defensive partner Irene Paredes, who spurned a glorious opportunity to nod wide of the Wolfsburg goal.
Barcelona dominated possession throughout the first period, but the German side demonstrated their clinical nature in front of goal to double their advantage before halftime.
Turning from scorer to provider, Pajor picked out Popp with an inch-perfect cross, allowing the Germany international to head home her 23rd competitive goal of the season.
Barcelona were seemingly down and out at the interval, but they flew out of the blocks to restore parity within five minutes of the restart
Caroline Graham Hansen's nimble footwork created the space to deliver a low cross to Guijarro, who was on hand to sweep the ball beyond Merle Frohms.
Within two minutes, Guijarro was celebrating her second, utilising a combination of head and shoulder to guide the ball home from Aitana Bonmati's lofted delivery.
After being blown away by Barcelona's electric start to the second period, Wolfsburg attempted to restore their lead through Pajor, who was denied by Sandra Panos on a couple of occasions.
Barcelona continued to carry the greater threat, though, and they subsequently completed the remarkable comeback in the 70th minute when Wolfsburg failed to clear their lines, presenting Rolfo with the chance to sidefoot the ball past Frohms.
Wolfsburg did their utmost to find a late equaliser, but Jonatan Giraldez's side held firm to win their third trophy of the season, adding to their league title and Spanish Super Cup.