The veteran struck a sublime free kick after Gonzalo Rodriguez was sent off to secure a quarterfinal spot for the Italian champions
The Italy veteran curled home a free kick to secure the victory for Antonio Conte's side after Gonzalo Rodriguez was sent off, meaning the Bianconeri remain on course to reach the final on home turf in Turin.
Juve beat Vincenzo Montella's men 1-0 in Serie A, but it was the hosts who held a potentially crucial away goal going into the game after a 1-1 draw in the first leg in Turin.
Fiorentina had dictated play for much of Thursday's encounter, but the last-16 tie turned midway through the second half when the Florence club saw skipper Gonzalo Rodriguez dismissed after conceding the crucial free kick on the edge of the area.
Pirlo capitalized on the opportunity, finding the top corner from the set piece to seal progress to the quarterfinals for Antonio Conte's men 2-1 on aggregate.
Carlos Tevez returned to the Juve starting XI after missing the first leg and the win over Genoa on Sunday, while Mario Gomez was preferred to Alessandro Matri at the head of the Fiorentina attack.
Fiorentina almost made the ideal start inside the first two minutes when Gomez – who scored the equalizer in the first encounter - raced through on goal, but the German could only scuff his shot wide.
Montella's men continued to look the more dangerous, David Pizarro's long-range drive forcing Juve goalkeeper Gianluigi Buffon into a fine reflex save just before the half-hour mark.
Buffon was the busier of the two keepers for much of the encounter and needed to be on his guard again to keep out a powerful swerving strike from Juan Cuadrado early in the second half.
Yet Juve remained a threat, spurning a glorious opportunity as Arturo Vidal headed narrowly over from Chilean compatriot Mauricio Isla.
However, Juve's hopes were significantly boosted on 69 minutes as Rodriguez was shown a second yellow card for a foul on Fernando Llorente.
And Conte's men immediately took advantage as Pirlo slammed the resulting free kick into the top corner beyond a helpless Neto.
From there Juve was able to take complete control of the contest and was denied a second when Neto produced a fantastic stop from Tevez's close-range header, but the Italian champions held on to maintain their hopes of reaching the final on home turf.