To fulfill the requirement of sample preparation in a microfluidic analysis system designed for "sample in, answer out" testing which was urgently needed by resource limited clinical facilities, we proposed a critical low cost, membrane-based serum separator design in this article. With a specially designed microchip, this device can easily separate serum from the whole blood sample in 5 min. Different from techniques which have been reported earlier, this approach does not require either centrifugation or sample dilution which may cause hemolysis or decreased testing sensitivity. By applying 300 μl of the whole blood sample, 50-70 μl of serum can be recovered from each device, and the serum volume recovery rate compared with centrifuged control is around 73% which is sufficient for most of the microfluidic-based assays. The protein recovery rate ranged from 70% to 95% which was compared with centrifuged control. The evaluation results indicate that this sample preparation device can offer sufficient amount of purified serum sample for any kind of diagnostic assays such as immunoassay and serum nucleic acid assay.