Juve are the runaway Serie A leaders and look certain to win the Scudetto once again, but Antonio Conte's men need to score at the Stadio Artemio Franchi after Fiorentina plundered a late goal through Mario Gomez to draw the first leg 1-1.
There is no love lost between the clubs from Turin and Florence, meaning Thursday's last-16 decider will have an extra dimension, and veteran midfielder Ambrosini hopes the "positive tension" in Tuscany will boost his side.
But as Juve continue to brush aside all comers in the league, the former AC Milan man is suitably wary.
He told violachannel.tv: "Are we the favourites? No. We would be crazy to think that.
"They're a team that inspire fear in opponents because they have a certain kind of spirit about them.
"They have players who are used to these kind of matches, from both a mental and technical point of view.
"There's a lot of positive tension around the city, and that will lift us, but the fans understand that we've got a long way to go before we can win.
"Anything can happen in such a finely-poised tie.
"These are games you have to face head on, with courage and paying close attention to what your opponent does."
La Viola are without wingers Ante Rebic and Joaquin, midfielder Mati Fernandez and strikers Giuseppe Rossi and Ahmed Hegazy through injury.
Coach Vincenzo Montella is expected to push attacking midfielder Josip Ilicic into the frontline alongside 20 million euro Germany star Mario Gomez, who scored only his third goal for the club in Sunday's victory over Chievo.
Juve, who beat Genoa 1-0 on Sunday to pull 17 points clear in the Serie A title race, have a serious crisis in defence.
Montella's counterpart Conte can only call on senior defenders Martin Caceres, Leonardo Bonucci and Giorgio Chiellini as Federico Peluso, Andrea Barzagli and Angelo Ogbonna struggle for condition.
Midfielders Claudio Marchisio and Sebastian Giovinco are unlikely to feature while strikers Carlos Tevez and Mirko Vucinic step up their recovery programmes following injury.
All being well, Tevez should start alongside Southampton loanee Dani Osvaldo in east Florence.
Centre-back Bonucci fired up his team-mates before the clash, telling the Gazzetta dello Sport: "On paper, our opponents have the advantage of playing at home, but only on paper.
"On the pitch we'll see which team deserves to progress.
"It will be a battle: may the best team win."
Juventus are more accustomed to life in the Champions League - winning the competition in 1996 and reaching the final on three occasions since - but they were losing finalists to Parma in the 1995 edition of the UEFA Cup.
Fiorentina, meanwhile, reached the UEFA Cup final in 1990, only to lose to Juventus.