Pulisic sustained a quad injury on international duty with the United States and is not available for Saturday's match, with Lukasz Piszczek and Maximilian Philipp also absent.
Dortmund are level on points with Bayern Munich at the top of the Bundesliga table, although the defending champions lead on goal difference ahead of the April 6 meeting between the sides at the Allianz Arena.
Favre, however, will have to do without Pulisic, who will join Chelsea ahead of the 2019-20 season, for the Wolfsburg clash.
"All of the players returned fit except for Christian Pulisic," Favre told reporters. "Unfortunately, he won't play on Saturday for sure.
"They are okay. They all took part in training. [Mario] Gotze, [Paco] Alcacer and [Axel] Witsel trained the whole week.
"Everything is fine. Still, Lukasz Piszczek and Maximilian Philipp are not ready for the match."
Pulisic scored a delightful goal for his country in their 1-1 draw against Chile, a cheeky chip over the goalkeeper after being put through on goal by Gyasi Zardes in the first half.
However, he was forced off soon after with injury, and now faces a race against time to be fit for Dortmund's crucial clash with Bayern.
Favre presser ahead of #BVBWOB:— Borussia Dortmund (@BlackYellow) March 28, 2019
"Wolfsburg is a very athletic team, They often change their formations, switching between a 4-3-3 and 4-4-2, we have to be prepared for everything." pic.twitter.com/3K9VKtfQwt
Gotze has been one of Dortmund's key men in their title charge and sporting director Michael Zorc confirmed the club are keen to offer the playmaker a new deal.
"We all realised that Mario Gotze is improving throughout the season and I hope he is not done yet. I hope he will keep improving," said Zorc.
"Regarding contract talks, we don't want to announce them officially but want to talk with the player personally."