Abstract: The single factor and orthogonal array design experiments of squid protamine extraction conditions,the basic nutrients of squid testes and the squid protamine were studied. The results showed that the yield of squid protamine was 3.42% in the optimal condition of 3 times volume of 0.3 M sulfuric acid concentration to extract protamine,repeating for 2 times,and 3 times volume of cold ethanol. The squid protamine contained 93% protein and was rich in all kinds of amino acids in which basic amino acids accounted for 26.2% and arginine accounted for 9.61%. According to the analysis of Tricine-SDS-PAGE electrophoresis,the molecular weight of squid protamine was less than 20 000 Da.