The current study focused on the pharmacodynamic activity components of Gentianopsis paludosa against ulcerative colitis (UC) fibrosis including symptoms of intestinal diarrhea and inflammatory. Trinitro-benzene-sulfonic acid induced UC model rats were gavaged with gradient polarity extracts respectively from ethanol-extract of Gentianopsis paludosa. Masson staining and qRT-PCR methods were respectively used to assess the degree of UC fibrosis and detect the mRNA expressions of collagen I, collagen III, a-smooth muscle actin (α-SMA) and E-cadherin in colon tissue. Separated by silica gel column chromatography, further screening was conducted until active components appeared. Infrared, nuclear magnetic resonance, mass spectroscopy and ultraviolet methods were applied to confirm active components’ structures. The results indicated that the expression of collagen I, collagen III and α-SMA mRNA in the colon tissues of acetidin group rats was obviously depressed compared with control groups while E-cadherin displayed just opposite. Dyed in blue indicating UC fibrosis degree, the area of acetidin group was less than that other experimental groups. Four components: (1,8-Dihydroxy-3,7-Dimethoxyxanthones, 1-hydroxy-3,7,8-Trimethoxyxanthones, 1,7-Dihydroxy-3,8-Dimethoxyxanthones and 1-hydroxy-3,7-Dimethoxyxanthones), were obtained from acetidin group and all of which have a significant equivalence to Gentianopsis paludosa on the therapeutic effect of UC fibrosis. Our findings revealed the activity components for clinical application history of Gentianopsis paludosa and provided a preliminary foundation for further new drug research and exploitation.