Abstract: This study aims to optimize the enzymatic hydrolysis process of anchovy and study the effect on proliferation of Streptococcus thermophiles by the anchovy hydrolysate.Basing on single factor experiments, the response surface methodology (RSM) was employed to optimize the enzymatic hydrolysiscondition of anchovy, by using proliferation effect on Streptococcus thermophilus growth as the evaluation index. The results showed that the optimum conditions were hydrolysis time 5.4 h, the ratio of raw material to water 1:2.2 (g/mL) and enzyme concentration 5.4%, and under these conditions, the ΔOD₆₀₀ of proliferated Streptococcus thermophilus was 0.556. The effect of different molecular weight segments on the proliferation of streptococcus thermophilus was studied, the results showed that the fragments with molecular weights of less than 3000 Da, significantly promoted the growth of Streptococcus thermophiles, moreover, the optimum content of the fragment in the growth medium was 1.2 g/L.The results of antioxidation experiments showed that the highest scavenging capacity of fragments with molecular weights of less than 3000 Da against DPPH⁺·was 70% respectively, and IC₅₀ value was about 2.73 mg/mL.