The hosts matched their opponents throughout the 90 minutes and were able to hold them to a goalless draw, bringing their run of four straight wins to a halt.
Juventus dropped points for just the third time in the league this season, as it was held to a 0-0 draw by Fiorentina on Friday night.
Massimiliano Allegri's men dictated play for much of the match on Friday, but failed to make the most of their superiority and subsequently handed second-placed Roma a boost in their battle to catch the champions.
The Juve coach seemingly had one eye on Tuesday's crucial Champions League clash with Atletico Madrid as he rested Carlos Tevez in favor of Kingsley Coman.
And the visitors seemed to miss the Argentinian's work rate and harrying in the final third, while clear-cut chances were few and far between.
Allegri eventually introduced Tevez from the bench with 30 minutes to go, but Fiorentina afforded the former Manchester City star little room for maneuver, generally nullifying his threat.
Vincenzo Montella's men were forced to withstand considerable pressure during the final stages, but held firm and claimed a commendable draw, meaning Roma can close to within one point of Juve with a win over Sassuolo on Saturday.
Juventus was greeted with a hostile atmosphere upon its emergence from the tunnel in the Artemio Franchi, with Fiorentina urged on from the start by its noisy support.
But Juventus appeared utterly unfazed as it put the hosts under pressure during the early exchanges, with Paul Pogba firing just over from the edge of the area in the third minute.
Fernando Llorente followed suit just three minutes later, as he turned away from David Pizarro and struck an effort agonizingly over the crossbar from 25 yards.
Fiorentina did begin to tighten up at the back, however, and after settling into the contest the hosts almost went ahead in the 32nd minute, as Matias Fernandez saw his curling free kick find the side-netting.
Borja Valero and Arturo Vidal both missed the target with speculative half-volleys for their respective sides in the final seven minutes of the first half.
The away side began the second half much like it did the first, taking just three minutes to craft the first opportunity, as Neto palmed away a Patrice Evra effort after Pogba's clever pass.
Juve looked more likely to break the deadlock, although Gianluigi Buffon found himself called into action with 66 minutes gone, parrying Juan Cuadrado's stinging drive from 25 yards.
Allegri's side poured men forward in the final 20 minutes, with Tevez's introduction appearing to galvanize the champions.
But the Argentine's initial influence quickly wore off, as both sides cancelled each other out and ultimately settled for a point after an unremarkable match.