Through a new experimental procedure, the influence of cross-flow velocity on the formation of fouling layer during microfiltration (MF) and ultrafiltration (UF) of biological suspension with 5 g/l of mixed liquor suspended solids was studied. Based on the resistance-in-series model, filtration resistances caused by adsorption, concentration polarization, and reversible and irreversible fouling were determined. Each filtration resistance was compared with respect to the mass, thickness and density of the corresponding fouling layer (or foulants). Moreover, the effect of fouling layer on permeate quality was investigated. The obtained results showed that permeate flux increased linearly with increasing cross-flow velocity and a high cross-flow velocity was more effective to reduce fouling of MF membrane than that of UF membrane. The formation of a reversible fouling layer was actually prevented by a cross-flow velocity of 3.0 m/s for MF membrane and 2.0 m/s for UF membrane. In all cases, the magnitude of reversible and irreversible filtration resistances matched well the mass and thickness of their corresponding fouling layer. Along with its mass and thickness, the density of fouling layer was another important factor affecting filtration resistance and dissolved organic carbon (DOC) rejection. Distinct fouling layers as well as pore blocking and the narrowed pore itself were the major contributions for enhanced DOC rejection.