Jones injured his ankle in the FA Cup win over Reading a fortnight ago and has not recovered sufficiently to face Madrid in Tuesday night's last-16 second-leg clash at Old Trafford.
When asked about the England international, Ferguson said: "He's not fit. He won't be fit for tomorrow."
Veteran midfielder Ryan Giggs will be involved, however, on what will be his 1,000th senior competitive appearance for club and country.
Ferguson added: "He played against QPR and I always think with Ryan that you like to keep him fresh.
"Tomorrow he will be involved at some point, whether starting or on the bench - I have not made my mind up but he will be involved. Of course his experience of big occasions will be vital to us."
Madrid come into the game on the back of two Clasico wins over arch-rivals Barcelona, the first of which saw them destroy the Catalans 3-1 at the Nou Camp.
Ferguson, whose side are currently 15 points clear at the top of the Barclays Premier League following Saturday's 4-0 victory over Norwich, said: "I think that both teams are in form.
"(They have given) two fantastic performances against Barca.
"I saw the first one and (they were) absolutely superb. They have found form at the right time for a game of this nature.
"Our form has been consistent for quite a while and we will need that form tomorrow night.
"They are two great clubs with great histories and it's set up to be a potentially marvellous game. I don't think it will be a disappointment."
Ferguson admitted the loss of Jones was a blow, given the England man's performance in the first leg, but was confident it was one his side could overcome.
"His performance over there was excellent but we do have options and hopefully it is something we get right," he said.
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