Abstract: The anaerobic-anoxic-oxic（AAO） process is a universal method for wastewater treatment and its secondary sedimentation tank activated sludge is rich in microbial resources. In this study, high-throughput sequencing was used to detect the structure and function of the microbial community in activated sludge from the secondary sedimentation tank, and the traditional isolation and culture method was used to screen the denitrifying phosphorus-accumulating bacteria. The results showed that bacteria in activated sludge of the secondary sedimentation tank were mainly composed of 23.2% Chloroflex, 20.6% Protecbacterna, 17.4% Actinobacteniota and 15.8% Bacteroidota. Among them, genera of norank＿f＿Saprospiraceae, norank＿o＿Saccharimonadales and norank＿f＿Caldilineaceae were dominant. The results of KEGG and COG annotation of partial gene showed that nitrogen metabolism, oxidative phosphorylation and phosphonate and phosphinate metabolism pathways existed in the sludge bacteria, and functional genes such as carbohydrate transport and metabolism, inorganic ion transport and metabolism were relatively abundant. Twenty-one species were isolated with R2A medium, belonging to Actinobacteniota, Proteobacteria and Firmicutes. Among them, there were 19 strains possessed the removal abilities for nitrogen and phosphorus, which were affiliated to Tsukamurella, Pantoea, Aeromonas, Staphylococcus, Acinetobacter, Pseudoxanthomonas, Gordonia, and so on. The strains Tsukamurella sp. D14, Pantoea sp. D6, Aeromonas sp. D8 and Gordonia sp. D4 had a nitrogen and phosphorus removal efficiency of higher than 80% for wastewater containing high concentrations of nitrate nitrogen（40 mg/L）, ammonia nitrogen（80 mg/L） and phosphorus（10 mg/L）. The rich microbial resources in activated sludge from the secondary sedimentation tank, especially the nitrogen and phosphorus removal bacteria were clarified further, which would provide a new basis and reference for the reutilization of the resource.