The Spanish champions will take on their Dutch counterparts at the Amsterdam ArenA and will hope to make it two wins out of two in Group D.
"I think there's enough motivation and if you ask my players I'm sure they will all say they are looking forward to playing this match," said the Portuguese.
"I have the ambition to win – it has always been like that in my career and always will be. I will prepare as always – as if it is my last match."
However, Mourinho is not underestimating the threat posed by Ajax.
"I prefer to look at Ajax as a team, they're much more important as a team than as individuals," he said. "We will try to be prepared to face their team.
"They know they're in the group considered by everyone as the toughest one because you have four champions from four football countries, which is unique."
Madrid won their first group game 3-2 against Manchester City at home last month.