Extracellular polymeric substances (EPS) are reported to be the major foulant in membrane bioreactor (MBR) processes. It is important to understand the EPS fractions which cause irreversible fouling to reduce operation cost and energy consumption in MBR. In this study, we developed polarity-molecular weight profiling, in which EPS components were plotted on two-dimensional matrix of its polarity and molecular weight, and applied it to investigate EPS of bulk sludge and cake layers from a labscale MBR. The EPS components were also characterized via three-dimensional excitation-emission matrix (3D-EEM) spectroscopy. The result showed that hydrophilic substances as large as 100 − 670 kDa was found only in loosely-bound EPS (LB-EPS) of bulk sludge but not in that of cake layers nor in permeate. Hydrophobic substances smaller than 20 kDa were mainly found in soluble microbial products (SMP) in bulk sludge. Hydrophilic substances larger than 670 kDa was mainly found in tightly-bound EPS (TB-EPS) of bulk sludge and in LB-and TB-EPS of cake layers. These findings suggest that, after conditioning of micropores of virgin membrane by hydrophobic substances smaller than 20 kDa in SMP, hydrophilic biopolymers as large as 100 − 670 kDa in bulk sludge clog the narrowed micropores inside membrane, causing irreversible fouling.