Midfielder Olmo, who helped Spain to European Championship glory last year, has been an integral part of Barcelona's team this season, while forward Victor, has been largely restricted to substitute appearances.
They had signed Olmo in August for around 55 million euros (£51 million) and Victor in July, but only registered them for the first half of the season due to La Liga wage cap considerations.
The Spanish Football Federation (RFEF) and La Liga said in a joint statement they had rejected a request to register them both for the second half, saying a cancelled licence could not be revived for a player by the same club in the same season.
"The Monitoring Committee agrees not to grant the prior approval or the definitive licence requested by FC Barcelona for the players Daniel Olmo Carvajal and Pau Victor Delgado," the RFEF said.
Two courts had previously rejected the club's requests.
La Liga had previously welcomed the legal rulings, saying additional spending is allowed so long-term injuries do not weaken a team but the rule should not be exploited to allow the registration of players whose salaries exceed the limit.
Spanish media said Barcelona plan to file a complaint to the Spanish government in a last-ditch attempt to allow the pair to play. There was no immediate comment from Barcelona.