The extraction and analysis of EPS in active biomass and membrane fouling were performed to investigate the influence of extracellular polymeric substances (EPS) on membrane fouling of MBR. The new membrane and fouling membrane surface morphology was characterized by EDX analysis. The results show that membrane contaminant is mainly composed of organic compounds. EPS in active biomass and membrane pollutant were extracted by method of alkaline regular centrifugation. Most of EPS were found to have high molecular weight over 12,000 D, which is very difficult to degrade. The total EPS contents in the extracted solution and the contents of polysaccharide protein and DNA in EPS were respectively analysed with TOC analysis and by phenol-sulfuric acid method, modification of the Bradford method and the fluorometric method. The analytical results indicate that the content of EPS in membrane fouling is about four times as much as that in active biomass, and polysaccharide is the most important pollutant on membrane, as well as protein's role in membrane fouling is not negligible, whereas the DNA is not important in membrane fouling because of its content (non-dialysis) in membrane contaminant being less than that in the active biomass. In addition, the results in electrophoresis experiment provide the information about the concentrated distribution of molecular weight of large fragment DNA in both membrane contaminant and the biomass.