On Monday, the Supreme Court declared all the actions of the assembly null and void until they stood down.
The three legislators say they agreed to leave the legislature to break the continuing stand-off.
Without the seats, the opposition loses a critical two-thirds majority.
The so-called "super-majority" would would allow the opposition to put a more effective challenge to the government of Nicolas Maduro.
They would be able to change the constitution and appoint new Supreme Court judges.
"We completely reject that ruling," said the three legislators on a letter that was read in parliament.
But their decision would "help free parliament from the institutional ambush they want to lead it into," they added.
The request by the three legislators - Julio Haron Ygarza, Nirma Guarulla and Romel Guzamana - was approved by the opposition-held assembly.
Members of the pro-government bloc celebrated the decision as "a victory for the people".
"We applaud this move, which puts the National Assembly back within the framework of legality," said pro-government legislator Hector Rodriguez.
The newly-elected National Assembly had defied the Supreme Court ruling, swearing in the suspended legislators last week.
Four lawmakers were barred by the Supreme Court - three from the opposition and one allied with the government - after the Socialist Party alleged irregularities during the 6 December vote for a new assembly.
The opposition claimed the the ruling was designed to strip it of the crucial two-thirds majority.
Without the four legislators, the opposition has 109 seats and the government 54. With the four banned legislators, the opposition gets the 112 seats it needs to reach the so-called "super-majority".
The four politicians are all from the rural and sparsely populated south-western state of Amazonas.
Venezuela's Supreme Court has almost always ruled in favour of the government during the last 17 years of socialist government under President Maduro and his predecessor, the late Hugo Chavez.
Three opposition politicians in Venezuela have agreed to give up their seats in the National Assembly while the electoral authorities investigate allegations of voting irregularities.