The trio did not pass fitness tests during today’s final training session at Milanello.
Thiago Silva limped off during Saturday’s 3-0 win over Palermo, while Seedorf and Ambrosini missed that match completely.
Also out of the 20-man squad named by Coach Massimiliano Allegri are Gennaro Gattuso, Alexandre Pato, Mathieu Flamini and the ineligible Pippo Inzaghi and Stephan El Shaarawy.
Kevin-Prince Boateng returns after he was suspended against Palermo, while Mario Yepes and Philippe Mexes are also passed fit.
Milan squad: Abbiati, Amelia, Abate, Antonini, Bonera, De Sciglio, Mexes, Nesta, Taiwo, Zambrotta, Yepes, Aquilani, Boateng, Emanuelson, Nocerino, Van Bommel, Cassano, Ibrahimovic, Robinho, Ganz