Abstract: Melanin is a type of natural pigment widely found in organisms and possesses various special functions. In this study, melanin was extracted from the ink of purpleback squid (Sthenoteuthis oualaniensis) by-product in the South China Sea, and its extraction process, physicochemical properties, and biological activities were systematically investigated to provide a reference for the preparation and application of this melanin. 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 iris sepia 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.