The development of a continuous process for cell separation is growing rapidly due to the current trend of cost-effective manufacturing in biological industries. The continuous cell separation process has a significant reduction in capital equipment costs and facility size compared to the conventional batch process. In the study, a multi-layered microfluidic-based device integrated with the porous membranes was fabricated for continuous size-based isolation of the cells based on the mechanism of restrictive cross-flow filtration, allowing the biological sample entered in a single inlet of the device and separated into two outlet streams. One stream which contained the cells returned back to the original sample fluid, while another stream with conditioned medium only was collected for later applications. The membrane fouling issue was overcome by introducing the alternative flow rate consisted of a set of higher and lower flows. The device integrated with the controllable flow restriction allows to increase the permeate flow rate, and alternative boosted flow demonstrates the high permeate flow rate (0.3 mL/min), high cell viability (> 98%), and increase of cell concentration (48%). As a result, we believe that the microfluidic-based continuous cell separation system is a promising tool for downstream bioprocess.