All three sides are still in the Liga title race, with Diego Simeone's side needing four points from their final two games to win the Primera Division.
Real Madrid need to win their final three games and will be hoping that Barcelona do them a favour when they tackle Atleti at Camp Nou and Ancelotti is certain that the Catalans will not take it easy.
"I'm sure that Barcelona will want to win," he said at a press conference.
"Barca will give their all against Atletico. They have always played like this and will continue to do so.
"It was an amazing end to the season because Levante had a very good match against Atletico despite playing for nothing as did Valencia, who were in the same sitution, against us.
"This is good for the league. We will all fight until the final minute. These three games are in our hands and we must win.
"[Wednesday's game against Valladolid] will be crucial because losing means not winning the league.
"Winning is the only option we have. The advantage is with Atletico because they need four points to become champions. We'll see what happens."
Ancelotti also ruled Gareth Bale out of the trip to Valladolid, but expects Cristiano Ronaldo to feature.
"Cristiano is fine, just a little tired. He just took part in training and is available at Valladolid. Carvajal, with slight discomfort, and Bale, who took a knock, are out."