The Argentinian will miss the remainder of the Premier League campaign with a knee injury but there had been hopes he would return for the second leg of the Round of 16 clash.
The 32-year-old was forced off just before half-time of City’s five-goal victory over Burnley in the Premier League last month following a forceful challenge from opposing defender Ben Mee.
The meniscus damage has a standard recovery period of between four and six weeks, meaning that a timetable of returning for the European game appeared feasible.
Guardiola was asked directly if Aguero would be available for the tie, as cited by Goal.com: “I told you many times no. No.”
City lead 2-1 from the first leg of the tie played in the Spanish capital back in February and it has confirmed that the return leg will be played at the Etihad in Manchester.
Should Aguero miss out against Madrid, Gabriel Jesus – who scored in the first leg – is likely to deputise.