Harrison has four fines this season totaling $125,000.
Harrison drew a roughing-the-passer penalty for hitting Fitzpatrick helmet-first during Pittsburgh's 19-16 overtime victory Sunday.
"James Harrison was fined $25,000 for roughing the passer, specifically he made helmet contact to the chest of the Buffalo quarterback, who was in a defenseless position at the time the contact was made," the NFL said in an e-mail Tuesday to The Associated Press.
After the game, the three-time Pro Bowl linebacker said the play didn't warrant a fine. He compared it to his Nov. 21 roughing-the-passer penalty against Raiders quarterback Jason Campbell that did not draw a fine.
"I'm not expecting a fine," Harrison said. "It's the exact thing as last week. It was the same exact hit. I hit him [Fitzpatrick] the same way I did the quarterback from Oakland and I got the same flag but I didn't get a fine. ... I'm expecting the same thing on this."
Harrison previously was fined $75,000 for a helmet hit on Browns wide receiver Mohamed Massaquoi, $20,000 for a helmet-to-the-back hit on Saints quarterback Drew Brees and $5,000 for a hit on Titans quarterback Vince Young.
On Monday, the league rejected Harrison's appeals of the $75,000 and $20,000 fines, although the Steelers supported Harrison's appeal.
Steelers coach Mike Tomlin declined Tuesday to debate whether Harrison should have been fined for the Fitzpatrick play. But Tomlin suggested Harrison needs to alter his tackling style to conform to the NFL's stricter enforcement of dangerous hits -- a toughened stance that was prompted, in part, by the Massaquoi hit.
"Obviously he does because the league doesn't agree with him," Tomlin said. "My opinion doesn't matter."
Tomlin also was asked if it's fair that the league keeps singling out Harrison for large fines.
"Man, life isn't fair," Tomlin said. "I'm not concerned about fairness. I'm really not. I'm concerned about preparing for the Baltimore Ravens" on Sunday.
Earlier this season, Tomlin and Steelers President Art Rooney II defended Harrison, and Tomlin told his players he doesn't want the league's stance to affect the way they play. Rooney also said the league was wrong for disciplining players for what he felt were legal hits, including the Harrison hit on Massaquoi.