Abstract: In order to optimize the enzymatic extraction process of chub mackerel immune active peptide, taking the proliferation rate and degree of hydrolysis of mouse spleen lymphocytes as indicators, on the basis of single factor tests, the enzyme addition amount, enzymatic hydrolysis time and enzymatic hydrolysis temperature were selected as the investigation factors, and the Box-Behnken method was used for three factors and three levels response surface test design to determine the best enzymatic preparation process of chub mackerel immune active peptide. The amino acid composition of the obtained immune active peptide of chub mackerel was analyzed. The results showed that neutral protease was the best protease, and the best enzymatic hydrolysis conditions were enzyme addition 8800 U/g, time 7 h and temperature 52 ℃. The verification experiment found that the relative proliferation rate of mouse spleen lymphocytes obtained under these conditions was 48.11%±2.67%, which was close to the predicted value of regression equation. At the same time, chub mackerel immune active peptide was rich in amino acids, including 45.52% essential amino acids and 32.00% hydrophobic amino acids. The results would provide a theoretical basis for enzymatic preparation of immune active peptide for chub mackerel, and a reference for further realization of high value utilization of chub mackerel resources.