The 23-year-old midfielder coolly finished off Alessandro Del Piero's delightful pass midway through the first half on a freezing Turin night.
"I must thank Alex, he's given me a splendid assist," Marchisio told Sky. "I'm so happy for this goal, finally my first. It is a very important result for the future."
Sixth-placed Fiorentina had several chances to equalise but Mario Santana's header hit the bar and Alberto Gilardino had a strike wrongly ruled out for offside before the fit-again Gianluigi Buffon saved the forward's flick.
Stevan Jovetic also had a strong appeal for a penalty rejected when he went down under Olof Mellberg's challenge.
Despite Fiorentina's pressure, the home side could have extended their lead with Del Piero again producing an exquisite through ball to release Marco Marchionni but the winger was twice denied by Sebastien Frey.
Juve, in their second season back in the top-flight after their 2006 demotion for match-fixing, have returned to the top on the sixth anniversary of the death of famous owner and Fiat head Gianni Agnelli.
Champions Inter, who lost 3-1 at Atalanta last weekend, can reclaim the lead outright on Sunday if they at least draw at home to Sampdoria. Top striker Zlatan Ibrahimovic is suspended for Jose Mourinho's side.
Third-placed AC Milan and Kaka, who rejected a move to Manchester City at the start of the week, travel to Bologna in Sunday's main programme of matches.
In Saturday's early game, Vincenzo Italiano smashed the ball home from distance in injury time to give Chievo a controversial 1-0 win at fellow Serie A strugglers Reggina.
The sides were joint-bottom before kickoff but Reggina now prop up the rest having conceded a goal moments after striker Bernardo Corradi had a second penalty appeal turned down when he was again manhandled by Chievo's Davide Mandelli.
Reggina also lost Carlos Carmona to a straight red card four minutes before the break when the midfielder aggressively thrust the ball into the chest of Chievo's Luca Rigoni.
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