The Amsterdammers open their campaign on Wednesday night against Lyon with De Boer already dismissing the possibility of his side winning the pool.
And with Lyon expected to be Ajax's closest challengers - Dinamo Zagreb are the rank outsiders - it is a crucial game for both sides.
De Boer told France Football: "It is very important because it's a big match in the race for second place.
"I think Real Madrid will finish on top but if we win tomorrow [Wednesday] it means we will take a step towards second."
Ajax have made a strong start to the defence of their Eredivisie title and currently lead the way in Holland once again having taken 13 points from five games.
De Boer has been encouraged by their recent performances and is relishing the prospect of seeing them compete in Europe.
He added: "I cannot wait to see how my young players will go when faced with a team which has more experience in this competition.
"This is just my second game as coach in the Champions League and I want to keep my unbeaten record."
Lyon boss Remi Garde agreed with De Boer in that the two sides are playing for the runners-up spot behind Jose Mourinho's Madrid.
"It would be very strange if Madrid did not finish on top," he said.
"We are competing with Ajax and Dinamo for second."
Lyon, like Ajax, have made a solid start domestically - Les Gones lie second having won three and drawn two of their opening five Ligue 1 fixtures.
Full-back Anthony Reveillere admits a new-look squad, with a new boss in Garde, is enjoying its football at the moment.
Reveillere said: "It was important to get a good start in the championship.
"It has been much better than in previous seasons. We have a new coach and a new playing staff.
"We write a new page and have many young players in the squad."