The scales is a kind of precious resource with high nutritional value. The collagen protein from scales have a widely usage prospection in the areas of health food, cosmetics and so on, and they are worthy of further studied. In this paper, the method of extracting collagen protein from the scales of Tilapia was studied by pepsin hydrolysis. First, the calcium was demineralized from the scales with using of 0.5 mol/L EDTA solution, then, the scales hydrolyzed by pepsin was made to extract the collagen, and the content of L – hydroxyproline was taken as an index to measure the extraction percentage. On this basis, the collagen extract conditions was discussed from the Tilapia scales by the pepsin. It was showed that the pepsin hydrolysis conditions were the enzyme dosage 3%, citric acid 12g/L, hydrolysis time 20h, so the rate of collagen extraction is higher.