The invention relates to a separation and purification technique of antioxidation dipeptide in protamine hydrolyzate, which includes six steps. Firstly, the protamine hydrolyzate is resolved in double distilled water, and supernatant is collected; secondly, the supernatant is sampled, components are collected and refrigerated in order to obtain lyophilized powder, which is resolved by double distilled water into solutions with different concentrations for measuring the scavenging activity of free radicals and separation and purification; thirdly, after separation and purification and elution,the eluted product is eluted by 20 mM Tris-HC1 buffer solution (pH 8.3) with a linear gradient of 0-0.35 M NaCl, and the eluted product is collected and refrigerated; fourthly, the lyophilized powderis resolved and then processed by a desalination column, product is collected and refrigerated; fifthly, the highest active components are separated and purified by a separation column and eluted by three percent of acetonitrile containing zero point one percent of trifluoroacetic acid, and the eluted product is tested under 280 nm, collected and refrigerated; sixthly, the highest active components are separated and eluted by a reversed phase column, and the eluted product is collected and refrigerated for measuring the scavenging activity and structure of free radicals. The invention can obviously scavenge the activity of hydroxyl free radicals and effectively reduce the content of the free radicals in MRC-5 cells, thus protecting cells form oxidative damage.