Abstract: This study aimed to optimize the preparation conditions of squid melanin enzymatic method by response surface methodology and analyzed its physicochemical characteristics.The squid ink sac was selected as the raw material,and the effects of pH,enzymatic hydrolysis temperature,enzyme dosage and enzymatic hydrolysis time on the yield of black carp were studied by single factor tests.Based on this data,Box-Behnken method was used to design the response surface analysis with four factors and three levels.The physicochemical characteristics of squid melanin were analyzed by UV spectroscopy,fourier transform infrared spectroscopy,scanning electron microscopy and X-ray diffraction analysis. Results showed that:The alkaline protease was selected to react with pH value of 10.40,enzymatic hydrolysis temperature of 65℃,volume of enzyme 2.60%(enzyme activity 2×10⁵ U/g),optimum enzymatic hydrolysis time of 2.45 h,the yield of squid melanin was 83.89%±0.21%. Ultraviolet absorption spectrum analysis showed that squid melanin prepared by alkaline protease enzymatic hydrolysis had characteristic absorption peaks at 201 nm. Strong absorption bands belonging to indoles ring structure appeared at 3388.46 and 1601.05,1373.66cm^-1 in fourier infrared absorption spectroscopy. Scanning electron microscopy results showed that the monomers of squid melanin were microspheric and the size distribution was concentrated,with an average diameter of about 120 nm.The results of X-ray diffraction spectroscopy could speculate the existence of indole structure of melanin,and the data support the Nano-aggregation model of melanin. The research results could provide theoretical basis and technical support for the high value development and large-scale production of squid melanin.