In order to utilize and control the invasive weed, Flaveria bidentis (L.) Kuntze, bioactive compounds mainly flavonoids from F. bidentis (L.) Kuntze were studied. High-speed counter-current chromatography (HSCCC) was successfully used for the separation of flavonol glycosides from F. bidentis (L.) Kuntze. The two-phase solvent system composed of ethyl acetate–methanol–water (10:0.4:10, v/v) was used for HSCCC. About 400 mg of the crude extract was separated by HSCCC, yielding 3.6 mg of patuletin-3-O-glucoside at a purity of over 97%; 4.4 mg of astragalin (kaempferol-3-O-glucoside) at a purity of over 98% and 4.5 mg of a mixture of hyperoside (quercetin-3-O-galactoside) and 6-methoxykaempferol-3-O-galactoside constituting over 97% of the fraction. The chemical structures were confirmed by MS and 1H, 13C, 1-D TOCSY NMR.