Objective The purpose of this study was to investigate the effect of total flavones of Desmodium triflorum, calycosin and formononetin from D. triflorum on mice with dextran sodium sulfate ( DSS)-induced ulcerative colitis (UC) and analyze the effects on intestinal flora. Methods Thirty C57BL/ 6 mice were treated with 3% DSS to establish the UC model and divided into the normal group, model group, total flavones of Desmodium triflorum group (500 mg / kg), calycosin group (100 mg / kg) and formononetin group (100 mg / kg). Body weight changes, disease activity index (DAI), colon histopathology score and IL-1β and IL-6 cytokine expression levels in the serum of the mice were determined, and changes of intestinal flora in mice were detected by high-throughput sequencing. Results Administration of total flavones of D. triflorum, calycosin and formononetin from D. triflorum effectively alleviated colonic inflammatory symptoms and reduced the expression of inflammatory factors in UC mice, Resulting in a good therapeutic effect. Treatment with total flavones of D. triflorum, calycosin and formononetin from D. triflorum significantly increased the diversity of intestinal microflora. The proportion of Helicobacteraceae was decreased and those of Muribaculaceae and Lachnospiraceae were increased at the family level. The proportion of Bacteroides was decreased and that of Lactobacillus was increased at the genus level. Regarding the species level, the proportions of Bacteroides and Escherichia coli were decreased. Conclusions Total flavones of D. triflorum, calycosin and formononetin from D. triflorum improve the changes of physical signs and inflammatory factors of UC mice and had a good recovery effect on intestinal flora.