Parliament will begin a debate on the motion on Thursday and a vote will be held within seven days.
"The prime minister ceases to hold his office after he has lost the confidence of this house," opposition leader Shehbaz Sharif said.
The vote comes as Pakistan faces an economic crisis, with Mr Khan's government banking on the International Monetary Fund to release the next tranche of a $6 billion rescue package to shore up dwindling foreign currency reserves.
Mr Khan, a former captain of the national cricket team, has been accused by the opposition of mismanaging the economy and foreign policy.
He is facing the most serious challenge to his leadership since coming to power in 2018.
After losing a parliamentary majority with a series of defections from his party, a united opposition is calling on him to step down. He has vowed to fight to remain prime minister.
The opposition needs a simple majority to topple Mr Khan, after which a new prime minister would be chosen by Parliament.
Nearly 20 politicians have defected from Mr Khan's party and his main coalition partners have suggested they could join the opposition, leaving him short of the 172 votes needed to hold on to power.
The opposition says it commands 163 votes and can obtain more.
“We have the support of required lawmakers to oust Imran Khan’s government,” said Bilawal Bhutto Zardari, the leader of the key opposition Pakistan People’s Party.
But Interior Minister Sheikh Rashid Ahmed said the no-confidence motion would be defeated.
The government has sought a court intervention to bar the defectors from voting against the party under an anti-defection law.
Loyal chief minister under threat
Opposition parties are also seeking to remove Mr Khan’s loyal chief minister in Punjab province, in a further blow to the embattled prime minister.
The opposition claims it has the backing of more than 20 defectors from the ruling party to succeed in the no-confidence vote in the provincial assembly.
But horse-trading is common in Pakistan politics and the group could return to the fold.
The opposition group moved a no-confidence motion against Chief Minister Usman Buzdar on Monday, said Samiullah Khan, a politician of Pakistan Muslim League-Nawaz, which leads the alliance of parties against Mr Khan.
The Punjab is the country’s largest province and a stronghold for Mr Khan's Tehreek-e-Insaf party, which has run the country for more than three years with a slim majority of about 12 politicians.
Thousands of Mr Khan's supporters gathered in the capital Islamabad on Sunday to rally behind him.
Political analysts say Mr Khan has lost the crucial support of the country's military.
Addressing the rally of supporters brought in on buses from around the country, Mr Khan said he was the subject of a "foreign conspiracy" aimed at dislodging his government.
"Funding is being channelled into Pakistan from abroad," he said.
"We have been threatened in writing but we will not compromise on national interests," he said, without offering evidence or details.
Police officials said up to 20,000 people attended the event.
There was a heavy security presence in Islamabad, opposition parties also planning rallies in the city next week.
Mr Khan was elected after promising to eradicate decades of entrenched corruption and cronyism, but the opposition has accused him of mounting a witch hunt against his opponents.
Voted in by an electorate weary of the two-party dynasties that had dominated Pakistan politics since independence, he has struggled to maintain support, with inflation rising, a currency crisis and crippling debt.