Abstract: Using cowhide as raw material, the process of ultrasonic-assisted enzyme extraction of collagen from cowhide was optimized. Based on the single factor experiment, a response surface test was designed, and the extraction rate of cowhide collagen was used as the response value to optimize the optimal extraction process of collagen, and its structure was characterized. The results showed that the optimal extraction conditions of collagen from cowhide were ultrasonic power 161 W, ultrasonic treatment time 64 min, pepsin addition 109 U/g, and solid-liquid ratio 1:16 g/mL. Under these conditions, the extraction rate of collagen was 63.77%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), ultraviolet spectroscopy (UV) and Fourier transform infrared spectroscopy (FTIR) analysis showed that the collagen extracted by ultrasound-assisted enzyme was in line with the characteristics of type I collagen, maintaining its complete triple helix structure, amino acid composition and scanning electron microscope (SEM) analysis showed that the collagen triple helix extracted by ultrasonic assisted enzyme was relatively stable. In this study, an effective process is optimized for the extraction of collagen from cowhide, besides, the extraction time of collagen is greatly shortened and it has high utilization value.