The collaboration featured at the 1992 Barcelona Olympics, after the Queen frontman’s death.
Mercury and the Spanish soprano, who starred in 90 opera roles and has been hailed for “inspiring millions” with her voice, first released the anthem in 1987.
The star, who had been in hospital since September, was known for her bel canto technique.
The Royal Opera House said it was “saddened” to hear of the death of Caballe, who was born into a working class family in Barcelona and was singing Bach cantatas at the age of seven.
“The Spanish soprano sang with us on a number of occasions between 1972 and 1992 and inspired millions over the course of her career with her stunning voice,” it said in a statement on Twitter.
Hospital Sant Pau spokesman Abraham del Moral confirmed Caballe’s death to the Associated Press.
The star was admitted to hospital because of a gall bladder problem, Spanish media said.