Di Matteo's biggest selection gamble since he took charge backfired as goals from Fabio Quagliarella, Arturo Vidal and Sebastian Giovinco left the Blues facing the prospect of becoming the first holders to depart the competition before Christmas.
Chelsea slumped to third place in Group E with one match remaining and must win against Nordsjaelland in two weeks' time and hope Shakhtar Donetsk beat Juve, despite the Ukrainian side having already qualified for the last 16 themselves.
If Chelsea's line-up was meant to ease the defensive jitters that had seen them fail to keep a clean sheet for nine matches, it took less than four minutes for them to re-emerge when they twice nearly gifted Juventus the lead, Stephan Lichtsteiner going close before Petr Cech somehow turned his volley onto the post.
The Blues were being penned in but almost scored a sensational breakaway goal in the ninth minute when Oscar ran 70 yards, beating three men before feeding Eden Hazard, whose shot ricocheted behind off Gianluigi Buffon's legs.
Oscar almost produced a goal out of nothing again when he raced onto Ramires' pass, Leonardo Bonucci getting back to block. And that proved crucial as Juventus took the lead in unconventional fashion seven minutes from half-time, Quagliarella reacting to redirect Anrea Pirlo's low drive past Cech, who could not keep the ball out having already moved to cover the original shot.
Forced onto the front foot, the visitors ended the first half on top but they were back to leaving far too much space in behind after the restart. They survived a penalty scare inside five minutes when Mirko Vucinic threw himself down under pressure from Gary Cahill and wasted a free-kick of their own when Juan Mata hit the wall.
Di Matteo took action by sending on Victor Moses for Cesar Azpilicueta on the hour but Chelsea were 2-0 behind a minute later in shambolic circumstances. Their defence was nowhere to be seen as Kwadwo Asamoah picked out Arturo Vidal, whose shot deflected off Ramires and through the admittedly luckless Cech's legs.
Lichtsteiner departed for Martin Caceres but there was no sign of a comeback as Juve threatened a third goal before Di Matteo finally turned to Fernando Torres, with John Obi Mikel making way.
Vucinic produced a Torres-like miss six yards out and Ramires was booked for sending Giorgio Chiellini sprawling. Chelsea tried to respond but Torres made no impression and Giovinco came off the bench to seal the game on the break, poking the ball beyond the onrushing Cech from 20 yards.