Abstract: Flavonoids have a variety of biological activities, and this work aims to provide basic data for the production and antioxidant application of flavonoids. Chaetomium globosum CGMCC 6882, an endophytic fungus isolated from Gynostemma pentaphyllum, was used in the present work to produce flavonoids.The fermentation process was optimized in terms of medium composition including carbon source, nitrogen source, trace elements, and inorganic salts, and fermentation conditions including fermentation time, inoculation amount, and pH value.The scavenging effects of flavonoids on DPPH free radicals, hydroxyl free radicals, ABTS free radicals, and superoxide anions as well as their effects on the activities of SOD, CAT, GSH-Px and the content of MDA were studied via in vitro and cellular antioxidant tests. The optimization results of fermentation process showed that the optimal media for flavonoid production by C.globosum CGMCC 6882 were starch, yeast powder, sodium molybdate, and dipotassium hydrogen phosphate; the optimal conditions were fermentation time 7.22 d, inoculation amount 4.88% (V/V) and pH 6.23. The antioxidant activity of flavonoids produced by C.globosum CGMCC 6882 showed a dose-dependent manner in vitro. The flavonoids produced by C.globosum CGMCC 6882 showed a positive effect on DPPH free radicals when the dose was 3.0 mg/mL, The scavenging rates of hydroxyl radicals, ABTS free radicals, and superoxide anions were (75.2±1.4)%, (84.3±0.8)%, (82.8±1.9)%, and (81.5±2.8)%, respectively. Cell oxidative damage model test results showed that the flavonoids obtained from C.globosum CGMCC 6882 can significantly increase the activities of SOD, CAT, GSH-Px enzymes in Caco-2 cells and reduce the content of MDA induced by H₂O₂ oxidative damage. The optimization process for C.globosum CGMCC 6882 fermentation successfully improved the production of flavonoids and the process was stable, which could be applied in industrial production. The flavonoids produced by C.globosum CGMCC 6882 showed promising antioxidant activity and can even protect cells from oxidative damage by reducing the activity content of MDA and enhancing the activities of several antioxidant enzymes. This work provided atheoretical guidance for microbial fermentation and application of flavonoids.