During the determination of molecular weight calibration curve of thymosin by size exclusion chromatography（SEC）,the acetonitrile ratio in mobile phase performanced significant influence on not only the retention time of four proteins（e.g.ribonuclease A,human insulin,thymosin α1 and somatostatin）,but also the linearity of molecular weight calibration curve.The best linearity was obtained at the ratio of acetonitrile by 75%,with a calibration equation of y=-3.138 6x+21.724 and a linear correlation coefficient(r2)of 0.988 5.In that condition,the theoretical plate numbers of four proteins were between 45 783 and 63 345,tailing factors were between 0.96 and 1.18,and resolutions were between 3.52 and 8.82.The established LC method to determine molecular weight calibration curve of thymosin by SEC was proved to have excellent separation and good linearity.Thus it can be used to detect the high molecular weight substance in thymosin preparation.