Abstract: In order to study the diversity of denitrifying bacteria in Hai river estuary, three sediment samples were collected from the freshwater, mixed-water and seawater environments of Hai River estuary and analyzed using clone library and canonical correspondence analysis (CCA). A total of 154 valid nirS sequences were grouped into 66 OTUs based on 97% sequence similarity. The diversity indices of those libraries showed the highest diversity of denitrifying community in the seawater sample. The phylogenetic analysis showed that the nirS sequences were divided into seven Clusters, which were highly similar to those of denitrifying bacteria from sediments of estuary, eutrophied bay, wetland and aquaculture wastewater. CCA indicated that salinity, organic carbon and nitrogen nutrient were the major contributing parameters for the diversity and spatial distribution of denitrifying bacteria of Hai river estuary.