The fire started overnight and had by morning encroached on the Doñana National Park at Moguer in the southern region of Andalusia, Jose Fiscal, deputy head of the regional environment protection authority, told Spanish television.
The reserve enjoys UNESCO protected status as an important biosphere reserve and is a tourist attraction. Many of those evacuated were staying at a camp site, Fiscal said.
The park is home to a wide variety of ecosystems and forms of wildlife including endangered species, notably the Iberian lynx and the Spanish imperial eagle.
Emergency services said local authorities sent in ten helicopters, seven planes and four waterbombing planes to deal with the blaze, which comes a week after forest fires killed more than 60 people in neighbouring Portugal.
Authorities were not able to speculate on the scale of the fire as it was still not under control by late morning.
The cause was also not clear and regional president Susana Diaz, who rushed to the area affected, indicated "the human factor cannot be excluded."
Spain was notably hit by a spate of forest fires in 2015 and officials said some of those could had been started deliberately.
At this time of year Spain is also having to battle drought and Sunday saw several areas classed as being at maximum risk of fire, including the Andalusian province of Huelva, where the Doñana Park is situated.