"She is a health professional who took care of the infected with the disease who were repatriated and cared for at Carlos III" hospital, the director of Spain's public health department, Mercedes Vinuesa, told a news conference.
Both the patients the nurse helped care for died from the disease.
Spanish priest Miguel Pajares, 75, was infected with Ebola in Liberia and died at Madrid's La Paz-Carlos III hospital on August 12.
Both were members of the Hospital Order of San Juan de Dios, a Roman Catholic group that runs a charity working with Ebola victims in Africa.
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The assistant nurse was admitted to hospital on Monday morning with a high fever, Spanish newspaper El Pais reported.
Doctors then isolated the emergency treatment room, the report said.
The Ebola virus causes fever, muscle pain, vomiting, diarrhoea and sometimes internal and external bleeding.
The current epidemic that has been ravaging west Africa is the worst outbreak of the disease yet, killing almost 3,500 people since the start of the year, with Liberia, Guinea and Sierra Leone worst hit.