Filtration characteristics and membrane fouling in BSA/dextran binary suspension cross-flow microfiltration is studied. An increase in cross-flow velocity or transmembrane pressure leads to higher pseudo-steady filtration flux due to less membrane fouling or higher driving force. The fouled membrane pore size and fouled layer thickness under various conditions are estimated using a theoretical model based on the Hagen–Poiseuille law. The fouled membrane pore size decreases with increasing transmembrane pressure due to ever increasing fouling. The fouled layer thickness becomes thicker and invariant with operating conditions under a lower pressure drop through the fouled membrane. Molecular adsorption occurs on the pore walls until a specific wall shear stress is reached in the membrane pores. However, the fouled layer becomes thinner when the pressure drop exceeds a critical value. An increase in cross-flow velocity results in a larger fouled membrane pore size and a thicker fouled layer in such condition. The dextran molecular deformation under higher transmembrane pressure causes a thinner fouled layer and an ultimate equilibrium adsorption in the membrane pores. Furthermore, an increase in cross-flow velocity leads to higher BSA and dextran rejection due to the sweeping effect on the membrane surface. The BSA rejection increases with transmembrane pressure due to the reduction in membrane pore size, while the decrease in dextran rejection under higher transmembrane pressure is attributed to the molecular deformation. The “coil-stretched” deformation of dextran molecules can be indicated using the Deborah number. The fouled membrane pore size and dextran rejection decrease with increasing Deborah number and remain constant as the Deborah number exceeds a critical value. Taking the filtration flux and solute rejection into consideration, operating a cross-flow microfiltration system under lower cross-flow velocity and higher transmembrane pressure is more efficient from the selectivity and mean dextran mass flux view points.