Catherine Ashton, the EU's foreign policy chief, welcomed the release of former Presidential candidate Andrei Sannikov and his campaign aide Dmitry Bandarenka.
"I call on the authorities of Belarus to release unconditionally now also all other remaining political prisoners and to remove all restrictions on the enjoyment of their civil and political rights. This would certainly contribute to possibilities for moving towards improved relations between the EU and Belarus," Ashton said in a statement.
Sannikov, a former deputy foreign minister and presidential candidate, was sentenced to five years of prison last year for taking part in a protest that followed Lukashenko's disputed re-election victory in December 2010.
On Saturday, Lukashenko said Sannikov had been released from his high-security prison thanks to a presidential pardon, a move that may help calm a diplomatic spat between Minsk and the European Union, which has been campaigning for his freedom.
"This is a result of solidarity, without solidarity there would not have been a decision [by Lukashenko]," Sannikov told Reuters by telephone from the city of Vitebsk.
"What I really want to do now is see my family... My (normal) prison term ended last September. Then torture began," he said.
Sannikaw is one of the leading opposition leaders in the tightly run authoritarian nation and his wife, a journalist, was also jailed by the authorities. She had already been released.
The EU had long demanded Sannikov's release, imposing travel bans and asset freezes on a number of Belarussian officials and businessmen to help secure his freedom.
That pressure triggered a diplomatic row with Minsk in February that led to all of the 27-nation bloc's ambassadors leaving Belarus.
Sannikov, 58, ran against Lukashenko in the 2010 poll which Western observers criticised as fraudulent. The vote handed Lukashenko, a former Soviet collective farm manager, a fourth term in office.
Sannikov had initially refused to ask Lukashenko for pardon but submitted such a request last December after what he described as threats against his family.
A number of other opposition politicians and activists were detained at the same rally and jailed afterwards, though most have since been released.
Lukashenko has run Belarus since 1994, tolerating little dissent and maintaining a Soviet-style economic system with artificially low prices, a large public sector and nearly full employment.
He has relied largely on financial support from Russia, which provides Belarus with cheap energy and other benefits, seeing it as a buffer between itself and NATO.
Human rights activists say about 15 people are still kept in Belarussian prisons on political grounds.