All trains between Madrid and Seville were cancelled after a rail employee noticed a suspicious package on the line, half an hour's journey from Madrid.
The latest alert comes as Spanish police continue to pursue suspected Islamic militants for last month's train bombings.
The Spanish army was ordered to protect the country's rail network yesterday after police defused a bomb on a track between Madrid and Seville.
The bomb, weighing 12kg, was connected to a detonation cable, rather than a mobile phone as in the previous attack, when 10 bombs claimed by al-Qaeda killed 191 people and injured more than 1,000 after exploding on four commuter trains in early rush hour traffic.
Three terror suspects blew themselves up as police in Madrid tried to arrest them in connection with the March 11 train bombings.
One police officer was killed in the blast and up to 15 people, including 11 officers, were injured.
Spain's Interior Minister Angel Acebes said the suspects fired shots before detonating a device when they were cornered in an apartment block.
Acebes said the four who died Saturday night included a Tunisian named Sarhane Ben Abdelmajid Fakhet, described by Spanish authorities as the leader of the group suspected of carrying out the March 11 train attacks that killed 191 people.