Mbappe has scored a club-record 213 goals for PSG during his six years at the club.
The 24-year-old Mbappe has so far refused to sign a contract extension with PSG, prompting speculation he could join the La Liga side.
It is believed PSG were open to selling the France captain rather than running the risk of losing him for free when his deal expires next year.
When asked about a deal for Mbappe, Ancelotti said "No, I rule it out 100%."
He added: "I think our squad is closed and our players are thinking about our season."
Mbappe spent a spell training away from the PSG first team this month amid his contract stand-off, but he has since returned following what the club described as "very constructive and positive talks".
He was left out of the club's opening game of the season against Lorient but scored a penalty after coming on as a substitute in PSG's 1-1 draw with Toulouse last week.
In July, PSG gave Saudi Arabian side Al-Hilal permission to talk to Mbappe after making a world-record £259m bid.
PSG chairman Nasser Al-Khelaifi has been adamant he would not let the striker leave for nothing and it is thought some kind of agreement is likely that will see Mbappe sign a one-year extension to his present deal as originally envisaged.
That would run to 2025 and allow PSG to sell Mbappe to Real Madrid next summer.
Mbappe joined PSG in 2017, initially on loan from Monaco before a 180m euro (£165.7m) transfer, and has scored a club record 213 goals in 261 games.
-- Courtesy of BBC Sport