Sane has enjoyed a strong start to life at Bayern since moving to Allianz Arena from Manchester City for €60 million (£55m/$70m) back in July.
The 24-year-old has contributed one goal and two assists in his first three appearances for the club, while also getting his hands on the UEFA Super Cup.
Bayern's victory over Sevilla at the Puskas Arena in Hungary last week saw them increase their haul of trophies in 2020 to four, and they will have the opportunity to add another piece of silverware to their collection when Dortmund arrive at the Allianz in midweek.
However, Flick's side head into that encounter still licking their wounds from a surprise 4-1 loss to Hoffenheim in the Bundesliga on Sunday, which ended their 32-game unbeaten run in all competitions.
The Bayern boss has now revealed that Sane will miss the showpiece event against Dortmund due to a knee injury, and that he will also be unavailable to feature for Germany during the upcoming international break, while David Alaba is a doubt with a muscle issue.
Flick told a pre-match press conference: "Alaba has muscular problems, so we will have to see how that develops. Leroy Sane will miss out. Hopefully, he will be back after the international break. He's got an injury in his knee."
🗣️ #Flick: "@David_Alaba has muscular problems, so we will have to see how that develops. @LeroySane19 will miss out. Hopefully he will be back after the international break. He's got a capsule injury in his knee."#FCBBVB pic.twitter.com/SprO8AmeLZ— FC Bayern English (@FCBayernEN) September 29, 2020
Asked to address Bayern's below-par showing against Hoffenheim, Flick responded: "My team's mentality is absolutely outstanding.
"Maybe one or two of them are slightly tired, but they always give 100 per cent on the pitch, which was also the case in Hoffenheim.
"Unfortunately, we didn't do some things that well that day."
He added on the challenge Dortmund will pose Bayern on Wednesday: "Dortmund have a fantastic young side and they also have experience in their ranks. They play attractive, offensive football, which I like.
"This will be a test against one of Germany's best. This game will help to develop us."
Bayern have been tipped to offload a few players before the transfer window closes, including Javi Martinez and Michael Cuisance, but Flick has insisted that both men remain a part of his plans for the time being.
"Both trained today, so I'm assuming they will be in the squad tomorrow," he said. "We all know that shortly before the window closes, the situation is not easy. We still have until October 5. Mika and Javi are still in the squad and are part of our team."