The sides drew 1-1 in the first leg of the last 16 tie in Spain, with Jones widely praised for his role in frustrating Cristiano Ronaldo at the Bernabeu.
But he has not featured since injuring his ankle in the 2-1 victory over Reading on 18 February and the England international has now lost his race to be fit.
When asked about Jones in Monday's pre-match press conference, Ferguson said: "He's not fit. He won't be fit for tomorrow.
"His performance over there was excellent but we do have options and hopefully it is something we get right."
Ryan Giggs looks almost certain to make his 1,000th career appearance at Old Trafford.
Ferguson opted to rest him in the 4-0 Premier League win over Norwich at the weekend and he now looks set to reach his landmark total in a high-profile contest.
The Scot 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."
Nani replaced Jones against Reading but may miss out to Shinji Kagawa in the battle for a midfield berth after he scored a hat-trick in the victory over Norwich.
Ferguson will also be able to call upon the experience of Rio Ferdinand. He was rested for the game against the Canaries but looks set to return against Jose Mourinho's side.
Ferdinand's fellow centre-back Jonny Evans trained on Monday but away from the rest of the squad, with the Northern Ireland international limited to light jogging.
Real, meanwhile, arrived in Manchester on Sunday night, with captain Iker Casillas travelling over with the rest of the squad before later being ruled out of the game.
Casillas has not played for Jose Mourinho's side since fracturing his left hand in their 5-0 win over Valencia in January and only returned to training on Thursday.
Spain's World Cup and double European Championship winner had wrestled back his starting place from replacement Antonio Adan before the injury but that setback forced Mourinho into signing fellow 31-year-old goalkeeper Diego Lopez from Sevilla.
Lopez looks likely to continue in goal in Manchester, with Real due to train at City's Etihad Stadium on Monday to continue preparations for the second leg.
Real and United drew 1-1 at the Bernabeu in the first leg after Cristiano Ronaldo cancelled out Danny Welbeck's early header.
Mourinho's men defeated arch rivals Barcelona 2-1 on Saturday afternoon, but are still 13 points behind the league leaders, meaning their main focus is on winning the European Cup for the first time in 11 years.