Abstract: In this paper, trypsin was treated with 100 mT magnetic field for 2 h, and the changes of trypsin activity and structure before and after magnetic field treatment were studied by infrared spectrum and circular dichroism. The antimicrobial activity of enzymatic hydrolysate produced by small yellow croaker hydrolyzed by trypsin was determined by inhibition zone method. The results showed that the enzymatic hydrolysate prepared by the enzyme treated with 100 mT magnetic field for 2 h had an increasing inhibition zone diameter, the inhibition zone diameter of Escherichia coli increased from (4.71±0.22) mm to (8.54±0.23) mm, and the enzyme activity increased by 52.5 U/min, compared with the untreated group. The change in the structure of trypsin was that the total amount of amino acids decreased, the proportion of essential amino acids increased, the peaks of the infrared spectrum were blue-shifted, and the proportion of α-helix and β-flod increased.