Abstract: Salty wastewater from seawater toilet-flushing in coastal cities can dramatically change the bioactivation and bioflocculation of activated sludge in sewage treatment system, particularly on extracellular osmotic pressure of microorganisms for the separation of cytodermand and cytoplasm. In order to improve bioflocculation for the removal of nitrogen and phosphorus, this study aims to investigate the effects of Fe3+ and Na+ on the removal of pollutants in activated sludge, and the evolution reaction mechanism of intracellular polymeric substances (IPS) and extracellular polymeric substances (EPS) in the anoxic zone of a A2O process. The contents of poly-β-hydroxybutyrate (PHB) and glycogen in IPS under multiple metal ions were analyzed using gas chromatography and anthrone colorimetry. The compositions and structures of EPS were characterized using three-dimensional excitation emission matrix (3D-EEM) fluorescence spectroscopy and fourier transform infrared spectroscopy (FTIR), in order to reveal the relationship between Fe3+/Na+, IPS, EPS and sludge flocculation. The results showed that the addition of single Fe3+ can contribute to the removal of COD, TN and TP, the activity of alkaline phosphatase and acid phosphatase, and the total IPS and EPS. More substances were accumulated on the surface of microbial cells under the flocculation of Fe3+. Compared with the addition of Fe3+ alone, the combination of Fe3+ (10 mg/L) and Na+ (0.5 g/L) can increase the removal of COD, TN and TP from 42%, 41% and 45% to 45%, 43% and 49%, respectively. Low concentration of Na+ can promote the respiration of microorganisms to save the generation time of cells, and thereby enhance the removal of organic compounds in activated sludge system. Although the increase in the total IPS and the activity of alkaline phosphatase and acid phosphatase, the low concentration of Na+ can inhibit the secretion of EPS to result in the decrease of the total EPS. The removal of COD, TN and TP decreased when Fe3+ was 10 mg/L and Na+ was higher than 1 g/L. In contrast, the high concentration of Na+ inhibited the microbial activity, and some microorganisms that cannot adapt to the environment were eliminated. The reason can be that the osmotic pressure outside the microbial cells increased to separate the cytoderm from the cytoplasm. The total IPS decreased, whereas the total EPS increased, indicating that the high concentration of Na+ can promote the EPS production. In FTIR analysis, the concentration changes of Fe3+ and Na+ did not cause significant changes of groups' compositions in LB-EPS and TB-EPS, where the main components were always protein (PN) and polysaccharide (PS). In 3D-EEM analysis, the addition of Fe3+ caused a visible-type tryptophan peak, while the addition of Na+ resulted in the peak degradation of tryptophan and humic acids, thereby to change EPS composition. A competitive growth was found between IPS and EPS. Specifically, when IPS/EPS was high (the accumulation of IPS was dominant, whereas that of EPS was relatively low), the bioflocculation of activated sludge was favorable. When IPS/EPS was low (EPS was relatively high), the bioflocculation can be slow in activated sludge during salty wastewater treatment system.