Background: Excessive reactive oxygen species (ROS) may overwhelm antioxidative defense, which is implicated in the onset and progression of various diseases. Flavonoids extracted from Penthorum chinense Pursh have a remarkable spectrum of bioactivities, while these pharmacological activities of flavonoids are at least partially due to their scavenging ROS and antioxidant ability. Therefore, we used the multicellular Caenorhabditis elegans as a model animal to investigate their antioxidant property and the possible molecular mechanisms in this study. Materials and Methods: The chemical compositions in P. chinense flavonoids were identified by ultra-performance liquid chromatography/time-of-flight mass spectrometry. Their anticancer and antioxidant ability in vitro were evaluated by inhibitory rate of HeLa cells, free radical scavenging, and iron-chelating ability. In vivo, synchronized L4 larvae C. elegans were treated by P. chinense flavonoids for 48 h. The survival rates of C. elegans were measured under heat stress condition. The antioxidant effect of P. chinense flavonoids on C. elegans was assessed by superoxide dismutase (SOD) and catalase (CAT) activities and malondialdehyde content. Results: P. chinense flavonoids was composed of six quercetin derivatives, two kaempferol derivatives, six pinocembrin derivatives, thonningianins A, and vanillic acid glucoside. P. chinense flavonoids not only showed a significantly inhibitory rate of HeLa cells but also exerted iron chelation and free radical scavenging ability. Furthermore, P. chinense flavonoids could extend the mean lifespan of C. elegans by approximately 17% under heat stress, which might be due to the increase of SOD and CAT activities. Conclusion: The study demonstrated that P. chinense flavonoids might be developed as a promising natural agent against environmental stress.