Alfredsson had been a game-time decision after participating in Ottawa's morning skate. Sens coach Paul MacLean said that Alfredsson passed a baseline test.
He was replaced in the lineup by Bobby Butler.
Alfredsson suffered a concussion in Game 2 on a hit from Rangers forward Carl Hagelin. Hagelin was also out Monday, serving the first game of a three-game suspension for the hit on Alfredsson.
NHL disciplinarian Brendan Shanahan called the hit, which occurred at 10:28 of the second period during the Senators' 3-2 overtime victory over the Rangers at Madison Square Garden, "a reckless strike to the side of Alfredsson's head."
Hagelin texted Alfredsson after the game and apologized for the hit. Hagelin also is Swedish and has said Alfredsson was one of his favorite players while growing up.
"I sent him a text yesterday that said I'm sorry and I'm very regretful for what happened," Hagelin said after Sunday's practice.
The Rangers released a statement stating they would not pursue an appeal, but said that they were "thoroughly perplexed in the ruling's inconsistency with other supplementary discipline decisions that have been made throughout this season and during the playoffs."
With Hagelin out, prized Rangers prospect Chris Kreider made his NHL debut as the Rangers took a 2-1 series lead with a 1-0 victory. Kreider, who signed with the team last week after the completion of his senior season at Boston College, was scratched for the first two games of the series.
Information from ESPNNewYork.com's Mike Mazzeo contributed to this report.