The Bianconeri reassured top spot in the Serie A table with a win over their fierce rivals after they were knocked off their perch by Udinese at the weekend.
Goals from Leonardo Bonucci and Alessandro Matri either side of a Stevan Jovetic equalizer fired the Bianconeri to their first win in three matches.
The victory sends Antonio Conte's men one point clear of Udinese at the league summit, albeit potentially temporarily, while maintaining their unbeaten start to the season.
Simone Pepe, after he was put in on goal by Mirko Vucinic’s intelligent reverse pass, and Claudio Marchisio both forced Artur Boruc into saves inside the first six minutes.
The hosts were in absolute control and even managed to waste two further opportunities, when Pepe made a fresh-air swipe at the ball and Vucinic side-footed wide, before taking the lead their dominance merited in the 13th minute.
Andrea Pirlo chopped a corner back towards Arturo Vidal, who swept a first-time short towards goal from 15 yards. Boruc blocked the effort but failed to hold onto the ball, and Bonucci demonstrated the type of poaching instincts associated with a striker by following up to force home the rebound.
The visitors were finding it impossible to stem the tide, and Boruc was forced into action on four further occasions, twice denying Vucinic before making impressive stops to thwart Pepe and Matri.
While the Old Lady would have been pleased with their first-half performance, they would have also been forgiven for being concerned by their inability to convert their absolute authority into goals.
And Fiorentina served them with a reminder about how precarious a one-goal lead can be in the first minute of the second half when Jovetic’s powerful downwards header from a Juan Vargas corner forced Marco Storari to push the ball over the crossbar.
However, Antonio Conte’s men failed to heed that warning, and after 10 minutes of concerted pressure, Fiorentina equalized.
A rapid raid saw halftime substitute Alberto Gilardino roll the ball to Vargas, who flicked into Jovetic’s path. The Montenegrin did the rest, curling a sumptuous effort into Storari’s top-left hand corner to restore parity.
The lead did not last long, though, as the Old Lady quickly edged back in front just eight minutes later. And Matri was the hero, slamming home from eight yards after Pepe had shown tremendous vision to prod the ball into his path through a gap in the Fiorentina defense, while also holding off the attentions of four opposition players.
The match continued to be played at a high tempo until the final whistle, but there would be no further scoring, ensuring that the Bianconeri would move back to the top of the pile until Wednesday evening at least.
Juventus returns to action on Saturday evening with a monumental Derby d’Italia clash against Inter at Giuseppe Meazza. Fiorentina, meanwhile, hosts Genoa on Sunday afternoon.