Jones had been a fitness doubt after injuring his ankle in the FA Cup win over Reading a fortnight ago, although manager Ferguson had attempted to give the England man every chance of making the last 16 second-leg clash at Old Trafford.
News of Jones's absence comes as a blow given his impressive performance against Cristiano Ronaldo and his Madrid team-mates in the first leg, which finished 1-1 at the Bernabeu.
Ferguson also said he is undecided over whether to start Ryan Giggs or bring him off the bench for his 1,000th senior appearance.
"Phil's not fit and won't be fit for tomorrow," Ferguson said. "Ryan will be involved at some point tomorrow, either as a starter or on the bench. I haven't made my mind up yet. He'll be important to us."
With Jonny Evans training on his own at the start of the session rather than involving himself in the normal keep-ball routines, Ferguson drafted in two reserve-team players to make up the numbers. Former Charlton trainee Sean McGinty and midfielder Luke Hendrie were the lucky players, but neither will feature against Madrid.
Instead Evans will compete with Rio Ferdinand and Nemanja Vidic for a place in the starting XI, with right-back Rafael asked to perform a marking job on Ronaldo.
"It's good to have that kind of problem as the three centre-backs are playing really well now," Ferguson said. "I'll make that decision tomorrow – hopefully it's the right one – but I couldn't be happier with the three of them."
Club director Avi Glazer was also present at training as the prelude to one of the most eagerly-anticipated European games stepped up a notch.
"It's a massive build-up," said Sir Alex. "You can see from the number of photographers at the raining ground that it's an indication of how important a game it is for both clubs.
"It's an occasion you always want to have and that's the beauty of the Champions League, you have the opportunity each year to play against one of the big teams from Europe. Tomorrow is one of the biggest."
Madrid come into the game on the back of two wins over arch-rivals Barcelona, the first of which saw them destroy the Catalans 3-1 at Camp Nou.
Ferguson, whose side are currently 15 points clear at the top of the 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."
United midfielder Michael Carrick acknowledges evenings like Tuesday night are the ones that really get his competitive juices flowing. After almost seven years at Old Trafford Carrick has enough experience not to be overwhelmed by the biggest of occasions. But he accepts certain matches are beyond the norm.
"These are the nights you live for," he said. "It is extra special. There is just something in the air. Everyone comes to the stadium with a spring in their step. Every supporter is that little bit more vocal. They appreciate how important it is and what it means to get through.
"The prestige and history of the club suggests we should be challenging in the later stages of these tournaments.
"It is up to us to go out there and do it."