Stephen O'Brien said the agencies had been told to leave by Friday, and several international non-governmental organisations (NGO) by Saturday.
The Luhansk separatists had earlier banned 10 international humanitarian agencies citing "violations".
Pro-Russian rebels seized parts of the Luhansk and Donetsk regions last year.
Almost 8,000 people have been killed since fighting erupted in eastern Ukraine in April 2014, a month after Russia annexed the southern Crimea peninsula.
In a statement, Mr O'Brien said the "de facto authorities" in Luhansk had ordered the UN agencies and NGOs to leave the area.
He also said all UN agency operations had been suspended in the rebel-held parts of Donetsk.
Mr O'Brien urged the separatists in "both Luhansk and Donetsk to ensure the immediate resumption of UN and international NGO activities".
He warned that restrictions on humanitarian shipments had prevented delivery of some 16,000 tons of vital supplies.
"Hospitals cannot perform surgery because they lack anaesthesia. Some 150,000 people are not receiving monthly food distributions," Mr O'Brien said.
On Thursday the Luhansk rebels refused to register 10 out of 11 foreign NGOs because of "violations".
They accused one of the banned NGOs - Doctors Without Borders - of "illegally storing psychotropic medication".
The NGO denies the allegation.
Ukraine and the West accuse Russia of arming the separatists and also sending its regular troops in eastern Ukraine.
Moscow denies this, but admits that Russian "volunteers" fight alongside the rebels.
The EU and the US have imposed their own sanctions against Russian officials and top allies of President Vladimir Putin.
A ceasefire in eastern Ukraine has been holding in the past two weeks, although there have been reports of occasional shelling.

Leah Washington, 18, and Joe Pugh, 19, both from Barnsley, were among four people badly injured on the Smiler ride on 2 June when two carriages crashed.
Ms Washington had a leg amputated and Mr Pugh suffered shattered knees.
The event takes place at the Metrodome Arena in Barnsley on 14 November and includes a pea and pies supper.
Midlands Air Ambulance, the Royal Stoke University Hospital and Staffordshire Fire and Rescue will benefit from the charity event.
Vicky Balch, 20, from Lancashire, also had to have a leg amputated and Daniel Thorpe, 27, from Buxton, Derbyshire, suffered serious leg injuries.
Twelve other people were trapped 25ft (7.6m) off the ground for several hours as emergency services attempted to rescue them.

The images appeared to show the pair on foot during a safari in June at the Gir National Park in western Gujarat state.
Officials at the park said they imposed a preliminary fine because "the cricketer flouted the rules".
Visitors to the park are not allowed to leave their vehicles.
Jadeja's photos were posted days after Gujarat forestry officials warned of the dangers involved in taking selfies with lions, following a number of attacks.
Wildlife officers say they will submit a final inquiry report to their superiors soon.
A.P Singh, chief forest conservator in Gujarat state, told AFP they "had instituted a probe and called Jadeja for a statement... but since he was not available, his father-in-law Hardevsinh Solanki gave a written statement on his behalf" and paid the fine for him.
Jadeja, 27, was part of India's team for the recent World T20 tournament.
A photo posted on Jadeja's Instagram account showed the cricketer pointing to a pride of lions behind him, with the caption, "Family photo, having good time in Sasan (Gir) #rajputboy #wearelions".
A second photo posted to the cricketer's account showed him and his wife, Reeva, with a lion looking on in the background.
The national park at Gir is the only place where Asiatic lions remain in the wild. The animals, which are slightly smaller than African lions, were listed as endangered in 2008.
The UN humanitarian aid chief has expressed alarm after UN agencies were ordered out of rebel-held parts of the Luhansk region in eastern Ukraine.
[NEXT_CONCEPT]
Two victims of the Alton Towers rollercoaster crash will hold a charity night to raise money for the organisations that helped them.
[NEXT_CONCEPT]
Indian cricketer Ravindra Jadeja has been fined 20,000 rupees (Â£229; $300) after posting photos online of him and his wife posing in front of endangered Asiatic lions.