Juventus dominated the league, bolstered by Cristiano Ronaldo who they purchased for 100 million euros (USD 112 million) last summer.
Juventus had been looking to reach the Champions League semi-finals for the third time in the past five seasons.
Juventus had needed just a point to claim their eighth Serie A title in a row with a record six games to spare, but suffered a shock defeat to lowly SPAL.
Cristiano Ronaldo had not played for his club since scoring a hat-trick to dump out Atletico Madrid in the last 16
Moise Kean came on as a substitute and fired in the winner on 84 minutes for his fifth goal in as many league games.
Cristiano Ronaldo hasn't played for the runaway Serie A leaders since scoring the sensational hat-trick that saw Juventus come back from a 2-0 first-leg deficit to knock out Atletico Madrid.
Juventus could win the league title with seven games to spare -- the earliest any team has ever sealed Serie A.
Moise Kean, 19, came on as a substitute in Turin and three minutes later scored his third league goal of the season.
Massimiliano Allegri said Cristiano Ronaldo is set to undergo tests on Monday to know exactly how long it will take him to fully recover.
Cristiano Ronaldo has denied allegations made by former model Kathryn Mayorga that he sexually assaulted her in Las Vegas in 2009.
Cristiano Ronaldo faces UEFA charges for "improper conduct" after mimicking Atletico Madrid coach Diego Simeone's gesture
Cristiano Ronaldo had made the contentious gesture in Juventus's 3-0 win over Atletico Madrid in the Champions League last Tuesday.
Cristiano Ronaldo was rested for the last six Portugal games while he settled at new club Juventus.
Cristiano Ronaldo's performance helped Massimiliano Allegri's men into the Champions League quarter-finals.
Cristiano Ronaldo has now scored 25 goals against Atletico Madrid in 33 appearances, including four hat-tricks.
Juventus' win over Napoli stretched their lead at the top of the Serie A to a huge 16 points.
Gareth Bale refused to celebrate his 78th-minute match-winning goal against Levante.