Almost 200 people have been infected since the virus was first detected at the city’s busy airport on 20 July.
All flights from Nanjing airport will be suspended until 11 August, according to local media.
Officials also began city-wide testing amid criticism for their “failure”.
All 9.3 million of the city’s residents – including those visiting – will be tested, said state-controlled Xinhua news.
Posts on social media show long lines of people queuing, and authorities have reportedly urged people to wear masks, stand one metre apart and avoid talking while they wait.
Officials said the highly contagious Delta variant of the virus was behind the infections, adding that cases had spread further because of how busy the airport is.
Ding Jie, a health official in Nanjing, told reporters the cases were linked to cleaners who worked on a flight from Russia that arrived in the city on 10 July.
The cleaners did not follow strict hygiene measures, Xinhua News reported.
The airport management has been rebuked, with a senior disciplinary body of the Communist Party saying it had “problems such as lack of supervision and unprofessional management”.
Testing has shown that the virus has now spread to at least 13 cities including Chengdu and the capital Beijing.
However, experts quoted by the Global Times said they believed the outbreak was still at an early stage and could be controlled.
Local officials in Nanjing said that seven of those infected were in critical condition.
The new spike in cases has led some on Chinese social media to speculate about whether the Chinese vaccines were working against the Delta variant.
It is unclear if those infected were vaccinated.
A number of South East Asian countries relying on Chinese vaccines have recently announced they will use other jabs.
China has so far managed to keep the virus largely under control by closing borders and moving quickly to stamp out local outbreaks.