The technology of extraction collagen from tilapia fish scales through pepsin hy- drolysis has been studied. According to hydrolysate concentration of hydroxyproline (HYP), the single factor and orthogonal experiment was determined the best enzymatic hydrolysis condition. The optimum condition of temperature, pH, enzyme concentration and hydrolyzing time for the ex- traction is 65~C, pH 2.0, 2%, 2 h. The extraction rate of collagen from tilapia fish scales was 94.24% under this optimal condition.