The unnamed female worker is the first person to catch the disease outside of Africa.
She was involved in the care of Fr Manuel Garcia Viejo in Carlos III hospital in Madrid when she became ill.
The priest picked up Ebola while working in Sierra Leone and was flown home to Spain for treatment before dying on 25th September.
Another Spanish priest, Fr Miguel Pajares, died in August after he contracted the illness in Liberia.
It is reported that the nurse was taken to hospital with a high fever on Monday morning and was tested for Ebola.
Her husband and 29 of her colleagues are now being tested.
So far, at least 3,400 people have died since the outbreak began, and there have been around 7,200 cases overall.