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With the increasing use of cupric oxide nanoparticles (CuO NPs), its potential environmental toxicity has been concerned nowadays. Aerobic granular sludge (AGS) is a special collection of microorganisms. This research studied under long exposure to the concentration of 5, 10 and 20 mg/L of CuO NPs, pollutants removal efficiency of AGS, extracellular polymers (EPS) and microbial communities in aerobic/anaerobic/anoxic (A/O/A) sequencing batch reactors (SBRs). The results showed that COD removal rates was stable, and the removal efficiencies of TN decreased because of the high concentration CuO NPs. On the 45th day, the TP removal efficiency of the reactor with CuO NPs concentration of 10 mg/L and 20 mg/L decreased to 55.83% and 43.72%, respectively. The denitrifying phosphorus removal-aerobic granular sludge (DPR-AGS) had certain resistance to the short-term impact of CuO NPs, and the phosphorus removal ability decreased at the late stage of the impact test. Besides, CuO NPs decreased the stability of DPR-AGS. High-throughput sequencing showed that CuO NPs decreased microbial diversity of DPR-AGS.