Flaveria bidentis (L.) Kuntze is a serious invasive plant with allelopathic properties in Hebei, China. To obtain the herbicidal substance from the root extracts of F. bidentis effectively and rapidly, the isolation and purification method was optimized by using high-speed counter-current chromatography (HSCCC). The results showed that the two-phase solvent system of nhexane-ethyl acetate-methanol-water (6:4:5:5, v/v/v/v) could separate the root extracts and get three components. The results of herbicidal activity screening for the three components indicated that component 3 had the strongest herbicidal activity against Digitaria sanguinalis and Arabidopsis thaliana. Component 3 was further separated and purified using highperformance liquid chromatography (HPLC) for structural determination, nuclear magnetic resonance (NMR) and mass spectrometry (MS) information validated the herbicidal componient 3 as α-terthienyl.