The separation performance is determined by the purification efficiency and the operation rate when membrane filtration is used for bio-separation. The Rotating-disk dynamic filter was designed and built in this case. Several hydrodynamic operating conditions, including rotation effect and feed flow rate, were used for reducing membrane fouling, increasing the permeating flux and improving the protein production. Furthermore, the distributions of fluid velocity and shear stress were simulated by using computational fluid dynamics. In shear-enhanced filtration system, the membrane fouling was mainly attributed to PMMA particle deposition on the membrane surface and in the membrane pores, respectively. Combined with shear-enhanced filtration experimental data concerning cake properties, such as thickness, porosity, and specific filtration resistance, the mechanism of cake formation based on a force balance model was analyzed. The effects of these operations on the filtration resistances, filtration fluxes and protein rejection were thoroughly discussed.