Morata suffered the injury during Tuesday's 2-0 defeat of Girona , which helped Atletico move eight points behind Barca ahead of Saturday's meeting between the top two at Camp Nou.
Diego Godin and Antoine Griezmann scored both the goals on the night to ensure Diego Simeone's team kept the pressure on the league leaders.
Atletico on Wednesday conducted tests on the Spain international, who had to be replaced by Juanfran after 85 minutes at the Wanda Metropolitano.
"The medical report indicates that [Morata] suffered an ankle sprain," said a club statement. "The striker is pending response to treatment."
Morata's absence would be a blow to head coach Simeone, who may also be without Diego Costa for the Barca game due to a hamstring injury.
Morata joined from Chelsea in the January transfer window, and has been a success at the side so far.
He has bagged four goals and one assist in eight starts in the league, helping to add some additional firepower to Atletico's attack.
But without Morata and Costa, the Madrid side may be forced to rely on Nikola Kalinic to supply offensive support to star man Antoinne Griezmann.
The 31-year-old has only scored four goals in all competitions this season, and is not a reliable goalscorer in the Spanish top flight despite being prolific in Serie A for Fiorentina, scoring 27 goals in two seasons there.
France midfielder Thomas Lemar is also another doubt due to a thigh complaint, with Atletico needing a win to boost their slim chances of catching the defending champions.
The two sides drew 1-1 earlier in the season back in November, with Ousmane Dembele's last-minute goal cancelling out Diego Costa's 77th-minute opener.
This time, Atletico desperately need a win over Ernesto Valverde's side to close to gap to just five points in La Liga with seven games left to play.
Barcelona dropped points on Tuesday in a thrilling 4-4 draw with Villarreal , with Luis Suarez scoring an injury-time equaliser to salvage a point.