Abstract: The research aims to use the enzymolysis technology to convert the insoluble protein from edible bird’s nest into water-soluble peptide, which could facilitate the digestion and absorption by the human body, and provide a foundation for the development of new products of edible bird’s nest. Using the hydrolyzing degree and the yield of peptide as index, alkaline protease was selected to hydrolyze the insoluble protein from edible bird’s nest. The effects of enzymolysis time, enzyme amount and substrate concentration on the yield of edible bird’s nest peptide were studied with single factor experiment. The response surface analysis was used to optimize the enzymatic conditions of the insoluble protein from edible bird’s nest. The optimal condition of enzymatic hydrolysis was: substrate concentration of 2.5%, the enzyme amount of 4%, enzymolysis time of 3.6 h, and the yield of peptide of 33.59%. The model was verified by the experiment: the yield of the peptide was 33.38%, and the relative error was 0.56%, which indicated that the model could well reflect the enzymatic hydrolysis of the insoluble protein from edible bird’s nest. In addition, gel electrophoresis experiments showed that the molecular mass of the insoluble protein was effectively reduced by using alkaline protease to hydrolyze the insoluble protein.