Investigation on the mass transfer behavior of multichannel flow electrophoresis (MFE) has confirmed the existence of concentration polarization in the separation process, which is characterized by a reduction of the relative protein migration flux corresponding to an increase in the protein concentration in the feed. The mobility of bovine serum albumin (BSA) in a gel membrane, as interpreted from its elution curve, was about one-sixth of its mobility in solution as determined by high capillary electrophoresis. This indicates that accumulation of charged protein onto the membrane surface occurs in MFE. An alternating electric field was applied instead of the steady one in MFE. The negative part of the alternating electric field reduced the polarization layer periodically. The effectiveness of this method was demonstrated by an over 40% increase of BSA migration flux at pH 6.9 in an optimized alternating electric field. The separation outputs of BSA and hemoglobin bovine blood conducted in an optimized alternating electric field at pH 6.0 were about 26 and 32% higher than their respective outputs obtained in a steady electric field. Because of its proven efficiency for reducing concentration polarization and its ease of operation, the use of an alternating electric field is a promising application in other membrane separation processes.