A rested Balotelli is set to lead Milan's attack alongside Alessandro Matri tonight.
The 23-year-old striker, Milan's leading scorer this season with four goals, returns after missing his team's last two Serie A games through suspension.
Balotelli was punished by the Italian league for insulting a referee during a 2-1 defeat to Napoli.
"In the past few days I've had a knee problem but I am ready to play against Ajax," said the former Manchester City striker.
"In Amsterdam, we want to give continuity to our victory over Celtic. Our aim is to arrive as calmly as possible to our double-header with Barcelona."
Milan won 2-0 against Celtic in their group opener and go into their midweek encounter inspired by Saturday's 1-0 triumph over Sampdoria, courtesy of Valter Birsa's goal.
Ajax, meanwhile, endured a dismal start to the group stages with a 4-0 defeat at Barcelona earlier this month.
The Dutch giants, whose last victory against a Serie A team in European competition was a 2-1 triumph over Roma 11 years ago, warmed up for tonight's game by demolishing Go Ahead Eagles 6-0 at the weekend. Ajax coach Frank de Boer was impressed by his team's display and expects more of the same when his side take on Milan.
"I wanted to see an Ajax that was aggressive in play, coaching, and fending off the opponent," De Boer said. "We showed passion and that is what we need to do on Tuesday."