The main objectives of this study were to investigate the influence of copper oxide nanoparticles (CuO NPs) on wastewater nutrient removal, bacterial community and molecular ecological network in activated sludge. The results showed that long-term exposure to 1 mg/L CuO NPs induced an increase of effluent concentrations of ammonia and total phosphorus, which was consistent with the inhibition of enzyme activities of ammonia monooxygenase, nitrite oxidoreductase, exopolyphosphatase, and polyphosphate in the presence of CuO NPs. MiSeq sequencing data indicated that CuO NPs significantly decreased the bacterial diversity and altered the overall bacterial community structure in activated sludge. Some genera involved in nitrogen and phosphorus removal, such as Nitrosomonas, Acinetobacter, and Pseudomonas decreased significantly. Molecular ecological network analysis showed that network interactions among different phylogenetic populations were markedly changed by CuO NPs. For example, β-Proteobacteria, playing an important role in nutrients removal, had less complex interactions in the presence of CuO NPs. These shifts of the abundance of related genera, together with the network interactions may be associated with the deterioration of ammonia and phosphorus removal. This study provides insights into our understanding of shifts in the bacteria community and their molecular ecological network under CuO NPs in activated sludge systems.