Abstract: Casein glycomacropeptide (CGMP) can be widely used in the function food and medicine due to its important physiological action. The authors studied the separation and purification conditions for CGMP from rennase casein hydrolysate by using Superdex 75 10/300 GL as media. The suitable conditions of separation and purification for CGMP are as follows: Superdex 75 10/300 GL is equilibrated and added samples and eluted with 0.1M ammonium acetate buffer, eluted velocity is 0.5ml/min, added volume of samples is 0.5ml when supernatant of casein hydrolysate is dewatered 25 times. The percent recovery of protein and the degree of glycosylation (sialic acid/protein) of CGMP obtained from the hydrolysate are 1.45% and 76μg/mg respectively. The CGMP contains three components whose molecular masses are 43000Da、30000Da and 25000Da respectively, and the degrees of glycosylation (sialic acid/protein) are 71.6μg/mg, 99.1μg/mg and 44.4μg/mg respectively.