In this study, we examined the long-term effects of copper oxide nanoparticles (CuO NPs) on the production and properties of EPS and the resulting variations in surface physicochemical characteristics of biofilms in a sequencing batch biofilm reactor. After exposure to 50 mg/L CuO NPs for 45 days, the protein (PRO) and polysaccharide (PS) contents in loosely bound EPS (LB-EPS) decreased as the production of LB-EPS decreased from 34.4 to 30 mg TOC/g EPS. However, the production of tightly bound EPS (TB-EPS) increased by 16.47 % as the PRO and PS contents increased. The content of humic-like substances (HS) increased significantly, becoming the predominant constituent in EPS with the presence of 50 mg/L CuO NPs. Furthermore, the results of three-dimensional excitation-emission fluorescence spectra confirmed the various changes in terms of the LB-EPS and TB-EPS contents after exposure to CuO NPs. Fourier transform infrared spectroscopy showed that the -OH and -NH2 groups of proteins in EPS were involved in the reaction with CuO NPs. Moreover, the chronic exposure to CuO NPs induced a negative impact on the flocculating efficiency of EPS and on the hydrophobicity and aggregation ability of microbial cells. The PRO/PS ratios of different EPS fractions were consistent with their hydrophobicities (R (2) > 0.98) and bioflocculating efficiencies (R (2) > 0.95); however, there was no correlation with aggregation ability. Additionally, the presence of bovine serum albumin (BSA) prevented the physical contact between CuO NPs and EPS as a result of NP aggregation and electrostatic repulsion.