A pilot-scale submerged membrane bioreactor (MBR) for real municipal wastewater treatment was operated for over one year in order to investigate extracellular polymeric substances (EPS) properties and their role in membrane fouling. The components and properties of bound EPS were examined by the evaluation of mean oxidation state (MOS) of organic carbons, Fourier transform infrared (FT-IR) spectroscopy, three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy, and gel filtration chromatography (GFC), etc. Test results showed that MOS of organic carbons in the bound EPS was ranging from -0.14 to -0.51, and major components could be assessed as proteins and carbohydrates. FT-IR analysis confirmed the presence of proteins and carbohydrates in the bound EPS. The organic substances with fluorescence characteristics in the bound EPS were identified as proteins, visible humic acid-like substances and fulvic acid-like substances by EEM technology. GFC analysis demonstrated that EPS had part of higher MW molecules and a broader MW distribution than the influent wastewater. It was also found that a high shear stress imposed on mixed liquor could result in the release of EPS, which would in turn influence membrane fouling in MBRs. Bound EPS solution was observed to have a stronger potential of fouling than mixed liquor. During long-term operation of the MBR, bound EPS demonstrated positive correlations with membrane fouling while temperature was verified as a negative factor affecting EPS concentration. Compared to tightly bound EPS (TB-EPS), loosely bound EPS (LB-EPS) showed more significant correlations with membrane fouling. This critical investigation would contribute towards a better understanding of the behavior, composition and fouling potential of EPS in MBR operation.