Membrane biofouling via microbial products limits the feasibility of utilizing membrane bioreactor (MBR) for treating wastewater. Fouling layer would be built up when activated sludge was filtered with a mixed cellulose ester membrane. This study probed the three-dimensional distributions of protein, alpha-polysaccharide, and beta-polysaccharide in fouling layer using fluorescently labeled lectins and fluorescein isothiocyanate (FITC) as staining agents in combination with confocal laser scanning microscopy (CLSM). These extracellular polymeric substances (EPS) distributed heterogeneously in the fouling layer, with alpha-polysaccharide being concentrated close to the membrane surface. The flow pattern yielded in the fouling layer determines the filtration resistance of biofouling.