Antler ossified tissue has been widely used for the extraction of bioactive peptides. In this study, collagen was prepared from antler ossified tissue via acetic acid and pepsin. Five different proteases were used to hydrolyze the collagen and the hydrolysate treated by neutrase (collagen peptide named ACP) showed the highest DPPH radical clearance rate. The extraction process of ACP was optimized by response surface methodology, and the optimal conditions were as follows: a temperature of 52 °C, a pH of 6.1, and an enzyme concentration of 3200 U/g, which resulted in the maximum DPPH clearance rate of 74.41 ± 0.48%. The peptides (ACP-3) with the strongest antioxidant activity were obtained after isolation and purification, and its DPPH free radical clearance rate was 90.58 ± 1.27%; at the same time, it exhibited good scavenging activity for ABTS, hydroxyl radical, and superoxide anion radical. The study investigated the protective effect of ACP-3 on oxidative damage in HaCaT cells. The findings revealed that all groups that received ACP-3 pretreatment exhibited increased activities of SOD, GSH-Px, and CAT compared to the model group. Furthermore, ACP-3 pretreatment reduced the levels of ROS and MDA in HaCaT cells subjected to H₂O₂-induced oxidative damage. These results suggest that collagen peptides derived from deer antler ossified tissue can effectively mitigate the oxidative damage caused by H₂O₂ in HaCaT cells, thereby providing a foundation for the utilization of collagen peptides in pharmaceuticals and cosmetics.