Objective To find out the optimal condition for separation of the protein mixture by gel filtration chromatography.Methods Separated the protein mixture of which known molecular relative weight by gel filtration chromatography in sephadex G 50,G 75 and G 100.Results The protein mixture were separated successful lyby gel filtration chromatography and the chromatogram of three separated samples were presented clearly.Conclusion Sephadex G 50 are suitable for purifying the large molecule protein,G 75 for the medium molecule protein,G 100 and G 200 for the small molecule protein.