Abstract: The preparation of microfluidic chip is investigated and improved in this research. Etching and bonding, which are key procedures in the microfluidic chip fabrication, were investigated and optimized. The effects of two kinds of etching formula were compared and the influences of etching time and the direction of oscillation during etching were studied. Moreover, it was found that the thermal bonding method with high temperature had longer lifetime than bonding under normal temperature. Using the new prepared chip, native protein electrophoresis has been realized. The increase of yield, the decreased of preparation efforts and the high performance of protein separation have been achieved by the improvement of chip preparation. This research exhibits good prospect of application in complicated biological separation.