The Australian World Cup winner had been an integral part of Oxford's plans, but he was powerless to prevent a 15-6 defeat in an error-strewn encounter.
Cambridge moved 59-52 ahead in the overall Varsity series thanks to a first-half penalty try and skipper John Blaikie's late close-range effort.
Roff said: "We really let ourselves down with our discipline.
"Cambridge played a really disciplined Varsity Match, and it is one of the most disappointing losses I have been associated with. Cambridge played better rugby on the day.
"We let ourselves down in certain aspects, although the effort was there, and there was a pretty sombre mood in the dressing room."
Roff, who scored 30 Test tries for the Wallabies in 86 appearances, saw little possession during a game dominated by Cambridge's watertight defence.
He produced glimpses of international class from outside centre, but superior Cambridge organisation sparked numerous errors by their opponents, including the game's pivotal moment - a penalty try.
Cambridge racked up 10 of their 15 points in the first half, yet they were never seriously threatened by a limited Oxford team.
Former Wasps back Jon Ufton converted the penalty try, and wing David Tibbott kicked a penalty to make it 10-6 at the break.
A 41,000 crowd though, had to wait until the last minute for the solitary second-half scoring contribution when Cambridge skipper John Blaikie crashed over from close range.
Blaikie said: "It wasn't the most pretty game to watch, but I would like to think we pleased the purists.
"We backed our defence today, and I think my try was probably my first for about five years. To win last year was amazing, but today was incredible."
Referee David Rose required assistance from video official Rob Debney before awarding the try that guaranteed Cambridge a deserved triumph highlighted by a superb display from back-row trio Nathan Johnson, David O'Brien and Ed Andrews.
Cambridge's former Wasps and Harlequins prop Jon Dawson collected man-of-the-match honours for his efforts in the scrummage battle, but Oxford trooped off reflecting on a case of what might have been.
Dawson said: "It was a wonderful occasion, and while it proved a dogfight, we really fronted up and we stuck in there."