Ronaldo, who has netted 14 goals in his last seven appearances for club and country, limped off at the start of the second half of last weekend's win at Almeria and a scan on Monday confirmed he had damaged a hamstring. (Agencies)
He missed Wednesday's Champions League victory at home to Galatasaray and, although further tests on Friday showed the muscle had healed, Ancelotti said the 28-year-old was not in shape to feature at the Bernabeu.
"No, Cristiano won't play because he is not comfortable and doesn't feel good," the Italian told a news conference.
"We did a scan this morning and while the muscle is completely healed he is not comfortable and hasn't trained much," he added.
"So we are not going to take the risk. He has 15 days to work and prepare well for the next league match (at Osasuna on December 14)."
Ronaldo is also likely to miss Real's King's Cup last-32 first leg at third-tier Olimpic de Xativa on December 7.
Ancelotti dismissed a suggestion his relationship with Sergio Ramos had soured after the Spain centre back was sent off for the 17th time in his Real career against Galatasaray.
Ancelotti criticised Ramos after the game but said on Friday there was "no problem" between the pair and he would play against Valladolid.
"It was simply a tactical discussion after the match," Ancelotti said.
"I think he made a mistake but that doesn't change anything. Making a mistake on the pitch is normal and it is important to understand why.
"Sergio Ramos is a very important player for this team and everyone knows that. He is fine, he has no problem, he will play tomorrow and hopefully will play well."
After 14 La Liga matches, third-placed Real are three points behind city rivals Atletico Madrid, who play at promoted Elche in Saturday's earlier kickoff (1500).
Atletico are a further three behind unbeaten leaders Barcelona, who are at fifth-placed Athletic Bilbao on Sunday (2000).
Ronaldo, who has netted 14 goals in his last seven appearances for club and country, limped off at the start of the second half of last weekend's win at Almeria and a scan on Monday confirmed he had damaged a hamstring.