As a typical flavonoid glycoside, swertisin mainly exists in sour Jujube seed. In this study, swertisin was extracted by ultrasound-assisted extraction method optimized with Box-Behnken design and response surface methodology. The antioxidant effect of swertisin was determined in vitro and in Caenorhabditis elegans (C. elegans). Furthermore, the potential mechanisms of its antioxidant stress were comprehensively evaluated and explored with network pharmacology and molecular docking technology. The results showed obvious scavenging ability of swertisin on free radical and swertisin (50, 250, and 500 μmol/L) significantly enhanced antioxidative enzymes activity (GST-4, SOD-3, and GSH-PX ) and reduced the reactive oxygen species and malondialdehyde accumulation in C. elegans, thereby protecting them from oxidative stress (heat stress and hydrogen peroxide). A total of 139 antioxidant targets of swertisin were screened and 70 signal pathways were enriched, including cancer-related pathways, lipid metabolism, liver injury-related pathways, acute lung injury, nervous system diseases, etc. This study provides the basis for further investigation on the antioxidant stress mechanism and contributes to the development of relevant drugs from natural products. PRACTICAL APPLICATIONS: The imbalance between the antioxidant defense system and reactive oxygen species is one of the main causes of neurodegenerative diseases, cardiovascular diseases, cancer, and aging. Therefore, alleviating oxidative stress injury has become a common strategy, which is helpful for the multi-target treatment of related diseases. The flavonoid of sour Jujube seed possesses potential antioxidant activity with multiple food health effects. From this study results, we optimized ultrasound-assisted extraction method for extracting the swertisin from sour Jujube seed and supported the use of C. elegans as an in vivo experimental model. We can recommend that the swertisin as a natural ingredient has a positive effect on antioxidation, which provided a scientific basis for treating related diseases through relevant pharmacological mechanisms and making antiaging functional food formula.