Using shark cartilage as raw material， the optimal enzymatic preparation process of collagen was optimized through single factor and response surface experiments， and the structure and stability of the prepared collagen were analyzed. The results showed that the optimal conditions of extraction collagen were as follows: 9 150 U/g of pepsin， 39.85 hours of extraction time， pH 1.67， and a ratio of 1∶15(g/mL). Under these conditions， the extraction rate of collagen was (85.09±0.17)%， and the prepared collagen retained a relatively complete triple helix structure， which conforms to the characteristics of type II collagen. The stability study showed that the denaturation temperature of the prepared collagen was 39.9 ℃， and its viscosity decreased significantly with the increase of temperature and salt concentration， but there was no linear relationship between the viscosity and pH value， and its viscosity was the smallest at pH 7 (4.26 mPa·s). Circular dichroism analysis further indicated that with the increase of salt concentration and temperature， the triple helix structure of collagen changed， when the temperature exceed the denaturation temperature of 39.9 ℃， the tripartite helix structure gradually unrotated and denatured. This study provides a specific theoretical basis for the high-value utilization of shark resources.