Samples collected from poultry stalls in a market in the Sham Shui Po area tested positive for the virus, said Hong Kong health secretary York Chow.
The government said all 2,700 birds at the market had been culled and the area sealed off Saturday.
Authorities raised the alert level to serious and urged people to seek medical advice if they had any flu symptoms.
Live poultry imports from China would be suspended for 21 days while an investigation was carried out, they said.
Chow said other markets were being checked and that culling would be extended if the bird flu virus was found in other locations.
Hong Kong was the scene of the world's first reported major bird flu outbreak among humans in 1997, when six people died.
H5N1 has killed more than 200 people and ravaged poultry flocks worldwide since 2003, according to the World Health Organisation.
Scientists fear the virus will eventually mutate into a form that is much more easily transmissible between humans, triggering a global pandemic.