This study aims to systematically identify the structure of an acidic homogeneous polysaccharide (USP-U) from Orchis chusua D. Don, and to evaluate its melanogenesis inhibitory and antioxidant activities. The structure of USP-U was elucidated through partial acid hydrolysis, Smith degradation, methylation analysis, and two-dimensional nuclear magnetic resonance spectroscopy. A total of 45 zebrafish larvae were randomly divided into five groups: the blank control group, the model group, the USP-U low-dose group (0.1 mg/mL), the USP-U medium-dose group (0.3 mg/mL), and the USP-U high-dose group (0.5 mg/mL). An oxidative stress zebrafish model was induced using 2,2'-azobis(2-methylpropionamidine) dihydrochloride to evaluate the protective effects of USP-U against oxidative stress in zebrafish. The results indicated that USP-U is primarily composed of mannose (Man) and glucose (Glc), and its proposed repeating unit is α-D-Manp-(1→4)-α-D-Glcp-(1→[4)-α-D-Manp-(1]₆→4)-α-D-Glcp-(1→6)-α-D-Manp-(1→. Results from the antioxidant activity assays revealed that USP-U exhibited protective effects in the oxidative stress zebrafish model, effectively reducing reactive oxygen species levels and cell mortality, and significantly ameliorated oxidative pathological damage in zebrafish by upregulating the activities of catalase (CAT) and glutathione peroxidase (GSH-Px), while lowering malondialdehyde (MDA) content. In conclusion, USP-U may serve as a potential antioxidant agent, providing a basis for the development and application of polysaccharides from O. chusua, and could also be utilized as a source of natural antioxidants.