Neither midfielder played any part in the 3-0 Serie A win against Chievo and they will not recover in time for Juve's opening Group D fixture.
Juve could also be without Giorgio Chiellini, with the defender struggling to recover from a calf injury in time for the trip to Camp Nou.
"Khedira and Marchisio are definitely out," Allegri told reporters. "Khedira returned from international duty, where he had a knock to the knee, rested for a few days and played 30 minutes.
"The day after, he felt pain again. Basically, it will take a week or so to have him back."
Mario Mandzukic came off with a knock against Chievo, but Allegri is hoping the Croatia striker and fullback Alex Sandro will be available.
"He has taken a calf on his calf, there should be nothing serious," Allegri said of Mandzukic. "Sandro had a fever, I hope he is already better."
Juve knocked Barca out of the Champions League at the quarter-final stage last season, winning 3-0 on aggregate, although they were later outclassed by Real Madrid in the Cardiff final.
And with Chiellini a doubt and Leonardo Bonucci having been sold to Serie A rivals AC Milan in a shock move, Andrea Barzagli could be the only remaining member of the 'BBC' defence that kept two clean sheets against Barcelona's feted 'MSN' attack.
"It's game of great prestige," said Barzagli. "Compared to five months ago, the stakes are different.
"Then it was a quarter-final and now it is a game for three important group stage points. It will be a tasty game, to show everyone that this year we want to move on."