The extended DLVO (XDLVO) theory was applied to elucidate the potential effects of CeO2 nanoparticles (CeO2 NPs) on sludge aggregation and the role of extracellular polymeric substances (EPS). In this study, seven different concentrations of CeO2 NPs were added to activated sludge cultured in sequencing batch reactors (SBRs) and compared with a control test that received no CeO2 NPs. After exposure to 50 mg/L CeO2 NPs, a negligible change (p>0.1) occurred in the sludge volume index (SVI), whereas the flocculability and aggregation of the sludge decreased by 18.8% and 11.2%, respectively, resulting in a high effluent turbidity. The XDLVO theory demonstrated that the adverse effects of the CeO2 NPs on sludge aggregation were due to an enhanced barrier energy. Compared to the van der Waals energies (WA) and the electric double layer (WR), the acid-base interaction (WAB) markedly changed for the various concentrations of CeO2 NPs. The EPS played a decisive role in the sludge surface characteristics, as the removal of EPS equals to the negative effects induced by 5–10 mg/L CeO2 NPs on the sludge flocculability and aggregation. The presence of CeO2 NPs induced negative contributions to the tight boundary EPS (TB-EPS) and core bacteria while positive contributions to the total interaction energy of the loose boundary EPS (LB-EPS).