The country has seen growing political tension and rallies, with three protesters dying in clashes last week.
An arrest warrant has been issued for opposition leader Leopoldo Lopez, who has pledged to lead a march in the capital Caracas on Tuesday.
The US has expressed concern about arrests of opposition protesters.
President Nicolas Maduro did not name the US officials being expelled, when he made the announcement in a national TV broadcast, but said the foreign ministry would give details later.
“It’s a group of US functionaries who are in the universities. We’ve been watching them having meetings in the private universities for two months. They work in visas,” the president said.
“Venezuela doesn’t take orders from anyone!” he added.
On Saturday, US Secretary of State John Kerry issued a statement expressing concern about the rising tensions in Venezuela.
“We are particularly alarmed by reports that the Venezuelan government has arrested or detained scores of anti-government protestors and issued an arrest warrant for opposition leader Leopoldo Lopez,” read the statement.
The main opposition grievances are high inflation, crime and the shortage of some staples.
The government has blamed the shortages on “saboteurs” and “profit-hungry corrupt businessmen”.
Opposition leader Leopoldo Lopez was last seen on Wednesday, when three men were shot dead at the end of opposition protests in the capital.
President Nicolas Maduro says an arrest warrant was issued against Leopoldo Lopez shortly after the incidents.
Nicolas Maduro has accused Leopoldo Lopez of inciting violence as part of a coup plot against his left-wing government.
The opposition says they were killed by pro-government militias known as “colectivos”.
Leopoldo Lopez, 42, is a former mayor of Chacao district, in eastern Caracas. He organized the recent protests against the government.
On Sunday morning, Venezuelan police searched the houses of Leopoldo Lopez and his parents.
Hours later, he posted a new message on Twitter and a three-minute long video. He said he had not committed any crime and challenged the authorities to arrest him at the next protest.
“I want to invite all of you to join me on a march on Tuesday from Venezuela Square [in central Caracas] towards the Justice Ministry building, which has become a symbol of repression, torture and lies,” Leopoldo Lopez said on the video.
He called on his supporters to dress in white, “to reaffirm our commitment to peace”.
“I will be there to show my face. I have nothing to fear. I have not committed any crime. If there is any order to illegally arrest me, well, I will be there,” added Leopoldo Lopez.
For his part, President Nicolas Maduro called on oil workers from the state company PDVSA to march to the presidential palace on Tuesday.
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