The composition and spectroscopic characteristics of extracellular polymeric substances (EPS) from activated sludge were analyzed in a sequencing batch reactor under Fe3O4 nanoparticles (Fe3O4 NPs) stress. Fe3O4 NPs promoted the activated sludge to secrete more total EPS, loosely bound EPS (LB-EPS) and tightly bound EPS (TBEPS) under long-term exposure. The protein and polysaccharide contents in the LB-EPS and TB-EPS displayed an increasing trend with the increase of Fe3O4 NPs concentration from 0 to 60 mg/L. The protein-like substances belonged to dominant fluorescence components in the EPS, whereas their peak location and intensity in the excitation-emission matrix fluorescence spectra had some evident changes under different Fe3O4 NPs concentrations. The groups of - OH and -NH2 branches of hydroxyl and amine groups in the LB-EPS and TB-EPs were sensitive to Fe3O4 NPs. The 0/C ratio in the LB-EPS and TB-EPS was more than the N/C ratio at each Fe3O4 NPs concentration, suggesting that the mass fraction of element was in the order of C > 0 > N.