In order to improve the comprehensive utilization of aquatic products and develop new functional foods containing probiotic bacteria, by-product of fish processing-the grass fish scales was studied. The grass fish scales were hydrolyzed by flavourzyme,and the enzymatic hydrolysis process was optimized by the single factor experiments and the response surface methodology (RSM) using the proliferation of Streptococcus thermophilus as the evaluation index. The results showed that the optimum conditions were the reaction time 3.8 h, the ratio of material to water 1∶3.2 g/mL and the concentration of enzyme 6.2%. Under these conditions,the ΔOD₆₀₀ of proliferated Streptococcus thermophilus was 1.173 4±0.015 0. Meantime, the GFSH under the optimum conditions was further separated into 5 segments by membrane filtration, which were:<1 ku,1-<3 ku,3-<5 ku,5-<10 ku,≥10 ku. The effects of five segments on the growth curve and production of acid of Streptococcus thermophilus were studied. The results showed that in the 24-hour growth period of Streptococcus thermophilus, the fragments with molecular weights of less than 1 000 u significantly promoted the growth and acid production of Streptococcus thermophilus, and the ΔOD₆₀₀ of proliferated Streptococcus thermophilus was 1.050,and the pH of the medium was reduced by 1.06.