Venezuela's move comes after EU foreign ministers agreed to sanction 19 Venezuelan officials a day earlier.
Venezuela has expelled the European Union's ambassador to Caracas, giving her 72 hours to leave the country, Foreign Minister Jorge Arreaza said, in a response to new EU sanctions against top officials.
"Today, by decision of President Nicolas Maduro, we presented to Mrs Isabel Brilhante ... her declaration as persona non grata," he told journalists in Caracas on Wednesday.
Venezuela's National Assembly, controlled by Maduro's party, called on Tuesday for the government to expel the ambassador a day after EU foreign ministers agreed to sanction 19 Venezuelan officials for "undermining democracy" and human rights abuses.
The move brought to 55 the total number of members of Maduro's regime targeted by asset freezes and travel bans by the bloc.
The EU expanded the list after rejecting a December legislative election that saw Maduro win total control of parliament after an opposition boycott.
The election results were not recognised by the EU, the United States or several Latin American countries.
Calls to reverse decision
Among the newly sanctioned officials are two members of the National Assembly, the governor of Zulia state, the commander of the armed forces and three members of Venezuela's electoral council, including its president.
Arrazea described the European measures as "illegal."
"Hopefully there will be reflection in the European Union, hopefully we will be able to rebuild the bridges of understanding, of dialogue, hopefully they will learn to respect," he said.
"We are doing this because the circumstances demand it," Arreaza added.
On July 29, after a previous round of European sanctions, Maduro had also declared Brilhante Pedrosa persona non grata and gave her 72 hours to leave the country. When the deadline passed, however, the government backed down.
"The EU profoundly regrets this decision, which will only lead to further international isolation of Venezuela. We call for this decision to be reversed," EU spokesperson Nabila Massrali said.
"Venezuela will only overcome its ongoing crisis through negotiation and dialogue, to which the EU is fully committed but which this decision undermines directly."