Abstract: Melanin is a natural pigment that widely exists in organisms and has many special functions. In this study, melanin was extracted from the by-product ink of purpleback squid (Sthenoteuthis oualaniensis) in the South China Sea, and absorbance was selected as the response value for response surface analysis. The preparation process, including enzymatic hydrolysis and extraction of melanin, was optimized. Its physical and chemical properties were investigated using UV, IR, solubility, and light and thermal stability tests. Additionally, its biological activities, such as photoprotection and antioxidant activity, were measured. The results show that the melanin from S. oualaniensis ink exhibited a characteristic absorption peak at 218 nm in the UV region. The optimal conditions for enzymatic hydrolysis of iris sepia ink melanin were as follows: pH 2, pepsin dosage 5 700 U·g⁻¹, hydrolysis temperature 45 ℃, and hydrolysis time 4 h. The infrared spectrum indicates that the main structure of S. oualaniensis ink melanin was an indole ring. This melanin was soluble in alkaline solutions but insoluble in organic solvents and water. It was prone to decomposition under high-temperature, natural light, and UV light conditions. However, melanin exhibited better protective effects against ultraviolet radiation and lipid peroxidation. These results provide a reference for the application of melanin from S. oualaniensis ink.