The new soybean stay-green variety Jinda Zhilv No. 1 (Z1) was obtained through crossing a stay-green mutant with the super-high yielding soybean cultivar Jinda No. 74 (JD74). Here, we compared the antioxidant enzyme activities and reactive oxygen species content of the Z1 and JD74 varieties under natural and dark-induced senescence. Dark treatment was imposed at the seedling stage for 13 days. Fluorescence quantitative PCR was used to investigate the expression of isozyme genes related to superoxide dismutase (SOD), catalase (CAT) and ascorbate–glutathione cycle. The results indicated that compared with JD74, Z1 exhibited enhanced antioxidant enzyme activity, with rates of hydrogen peroxide and superoxide anion accumulation being lower in Z1 after flowering. The expression levels of antioxidant enzyme isogenes, including Mn-SOD, Chl Cu/Zn-SOD, peroxisome Cu/Zn-SOD, CAT5, MDHAR1, and DHAR3, were higher in Z1 than in JD74 during the seed-filling stage. After 6 days of dark treatment, the membrane system of JD74 leaves showed severe oxidative damage and the leaves had turned completely yellow. These changes were accompanied by reduced contents of chlorophyll and soluble protein after 13 days of dark treatment. In contrast, Z1 was observed to be more tolerant to dark stress. Its internal reactive oxygen metabolism balance remained unimpaired, and the leaves showed no obvious senescence traits. In conclusion, the higher antioxidant capacity in Z1 contributes to delayed leaf senescence, which is a significant finding with respect to the application of stay-green mutants in soybean breeding and germplasm innovation. © 2021 Friends Science Publishers