By using a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (2:2:2:2, v/v/v/v), a high-speed counter-current chromatography technique was successfully used for isolation and purification of three alkaloids from Picrasma quassiodes (D. Don) Benn. for the first time. A total of 22.1 mg of 3-methylcanthin-2,6-dione, 4.9 mg of 4-methoxy-5-hydroxycanthin-6-one and 1.2 mg of 1-mthoxycarbonyl-β-carboline were obtained from 100 mg of crude extract of Picrasma quassiodes (D. Don) Benn. in less than 5 h, with purities of 89.30%, 98.32% and 98.19%, respectively. The target compounds were identified by ESI-MS, (1)H NMR and (13)C NMR.