A pilot-scale hybrid membrane bioreactor (HMBR) for real municipal wastewater treatment was developed by adding biofilm carriers into a conventional membrane bioreactor, distribution and dynamic changes of the extracellular polymeric substances (EPS) and their roles in membrane fouling were investigated. The results showed that the concentrations of loosely bond EPS (LB-EPS) and tightly bond EPS (TB-EPS) in activated sludge, carrier biofilm and sludge cake layer have been increased significantly with the running time of HMBR, during operation of the HMBR, EPS demonstrated positive correlations with membrane fouling. Compared to TB-EPS, LB-EPS showed more significant correlations with sludge physical properties and specific resistance to filtration (SRF) in HMBR, and thus demonstrated that LP-EPS have a stronger potential of fouling than TB-EPS. It was also found that a lower organic loading in HMBR could result a significant increase in EPS concentration, which would in turn influence membrane fouling in HMBR. This critical investigation would contribute towards a better understanding of the behavior, composition and fouling potential of EPS in HMBR operation.