Ulcerative colitis (UC), an immune system disease, is characterized by long duration and easy relapse. Sophora flavescens (S. flavescens), also named "Kushen", is a traditional Chinese medicine, widely used to treat UC in clinics. Alkaloids and flavonoids are the main constituents of S. flavescens. Previous studies indicated that the effects of S. flavescens against UC mainly attribute to its alkaloids. In view of the clinical applications of its flavonoids and our preliminary experiments on the effects of S. flavescens treatment, we speculated that flavonoids also could exert an anti-UC effect, but its efficacy and mechanism are still not yet to be revealed. Herein, we examined the pharmacodynamic effects of the ethyl acetate (EtOAc) extract of S. flavescens EtOAc (SFE) against dextran sodium sulfate-induced UC rats for the first time. Pharmacodynamic effects indicated that SFE could significantly alleviate the loss in the body weight and shortening of the colon length, reduce colon bleeding and improve colon tissue damage of UC rats. A total of 28 prototypes and 41 metabolites were unambiguously or tentatively detected in rat's plasma and urine. Among them, 28 prototypes and 3 phase I metabolites shared 40 UC targets, the targets contributed to 51 metabolic pathways in 5 modules. Additionally, genistein, formononetin, isokurarinone, kurarinone, maackiain, kushenol N, trifolirnizin, kuraridin and norkurarinone were suggested to be potential active compounds in SFE for treating UC by comprehensively investigating the results of network pharmacology analysis, metabolic analysis in vivo, and previous researches. Finally, a combination of metabolic analysis in vivo with network pharmacology can elucidate the material basis and pharmacodynamic effect of traditional Chinese medicines, and lay the foundation for further clarify the anti-UC mechanism of SFE.