In order to investigate the protective effect of Sepia esculenta ink melanin on the oxidative damage of human cloned colon adenocarcinoma cells (Caco-2), the ultrasound-assisted method was used to prepare Sepia esculenta ink melanin and its structure characterization and antioxidant capacity were measured. In this study, 150 μmol/L tert-butyl hydrogen peroxide (TBHP) was used to induce the oxidative damage model of Caco-2 cells, and the cell survival rate was determined by the MTT method, as well as the activity of superoxide dismutase (SOD) and malondialdehyde (MDA) in cells were determined. The experimental results showed that the melanin extracted by ultrasound retained the characteristic group and indole structure, and the particle size became smaller. Compared with the oxidative damage model group, the melanin group can effectively improve the survival rate of damaged cells. The interference of S. esculenta ink melanin with concentration above 40 mg/mL could effectively increase the activity of SOD and reduce the level of MDA in cells, and protect Caco-2 cells from the damage caused by oxidative stress.