Abstract: Casein was hydrolyzed by neutral protease, alkaline protease and trypsin to determine the best protease for preparation of cholesterol-lowering peptide. The effects of hydrolysis pH value, hydrolysis temperature, enzyme to substrate ratio, substrate concentration and hydrolysis time on casein hydrolysis degree and cholesterol micelle solubility inhibition rate were investigated by single factor and response surface experiments, and the optimal hydrolysis conditions were determined. Then the separation process of cholesterol-lowering peptide was determined by ultrafiltration and gel filtration chromatography. The results showed that the optimal enzyme was neutral protease, and the optimal hydrolysis conditions were as follows: Reaction temperature 51.3 ℃, enzyme to substrate concentration ratio 6.47%, pH6.34, substrate concentration 5 g/100 mL, reaction time 3.5 h , and cholesterol inhibition rate 58.25%±0.59%; The separation conditions of casein cholesterol-lowering peptide by Sephadex G-10 were as follows: Loading concentration 80 mg/mL, loading volume 2.5 mL, elution rate 3.5 mL/min; The inhibition rates of cholesterol solubility of peak 1 and peak 2 samples were 24.2%±0.24% and 4.3%±0.16% at 100 µg/mL after ultrafiltration and chromatography.