Goals from Neymar and Alexis Sanchez secured Barcelona the points, with Real scoring a late consolation through substitute Jese.
However, Ancelotti was adamant Javier Mascherano's challenge on Cristiano Ronaldo with 19 minutes to go when the score was 1-0 to Barcelona was a penalty.
Referee Alberto Undiano Mallenco waved play on after Ronaldo tumbled under the Argentinian's challenge, to the fury of Ancelotti and the Real substitutes.
"Naturally the penalty was very clear for me. I think everyone saw it, the only person that didn't see it was the referee," Ancelotti said in the post-match press conference.
Real's players also thought they could have been awarded a spot-kick towards the end of the first half for a handball by Barca left-back Adriano.
The Brazilian had already been booked before the incident and, although Ancelotti was less certain about the handball, he reflected: "It's a difficult decision, but it was at an important moment and if the penalty had been given, it may have been another yellow card too."
Real captain Sergio Ramos, who was deployed in the unfamiliar position of central midfield, was more disparaging about Mallenco's decisions.
The defender told reporters: "The team fought toe to toe against a very difficult team, and by the end we had them on the ropes, it's rare that you see Barcelona trying to waste time at the end of a game.
"But there were two clear penalties and, although I don't like to try and justify defeats by talking about referees, there are some things you cannot fight against."
Ancelotti explained that he played Ramos in midfield because Asier Illarramendi had a muscle strain following the 2-1 win over Juventus in the Champions League on Wednesday, and defended the decision to field the Spain international alongside Luka Modric and Sami Khedira in midfield, a cautious tactic despite promising before the match to take the game to Barca.
He said: "I played Ramos there to look for balance in the middle of the park, I asked him about it before and he said he wanted to play there, so I put him in."
He added: "I didn't play defensively, Modric isn't a defensive midfielder, and Marcelo, (Dani) Carvajal and Cristiano and (Angel) Di Maria are attackers. Barca did not play with any more attackers than we did."
Reflecting on what was his first experience of a Clascio, the Italian said: "It's a fantastic game and atmosphere. I think it was a good game, we showed we can play well against a fantastic team. Of course you want to win this kind of game but at the moment we are improving and this game proves that."