One new flavanonol 4 H-​1-​benzopyran-​4-​one,2-​(4-​hydroxyphenyl)​-​3,​7-​dihydroxy-​5-​methoxy-​8-​[5-​methyl-​2-​(1-​methylethenyl)​-​4-​hexenyl]​(21), and twenty-six known compounds 1–20, 22–27 were isolated from the dried root of Sophora flavescens (S. flavescens) in this chemical study. Their structures were elucidated according to the spectroscopic and spectrometric methods. All the isolated compounds were evaluated for their cytotoxicity against lung cancer A549 cells and colon cancer HCT116 cells. Among them, compound 21 showed relatively predominant cell proliferation inhibition effect on the two tumor cell lines. Moreover, it induced cells apoptosis as evidenced by the Annexin V/PI double staining assay as well as the increased cleaved-PARP expression. The aforementioned data indicated that the flavonoids of S. flavescens have potential anti-cancer effect.