Abstract: The aim of this study was to develop a method for the separation and preparation of high purity sanguinarine and chelerythrine by conventional high speed counter-current chromatography. After comparing six kinds of solvent protocols of analytical high speed counter-current chromatography (HSCCC), the two phase system of chloroform-methanol-0.2 mol/L hydrochloric acid aqueous solution (4:2:2,V/V/V) was finally chosen as the operating solvent of preparative high speed counter-current chromatography (HSCCC), in which the upper phase was used as the stationary phase and the lower phase was used as the mobile phase. Under the conditions of ratations speed of 455 r/min, lower phase flow rate of 8 mL/min, the injection volume of 1000 mg, the temperature of 25 ℃, sanguinarine and chelerythrine was separated and preparated on HSCCC. The results showed that 505 mg of sanguinarine chloride and 435 mg of chelerythrine chloride could be obtained from 1000 mg of crude Macleaya cordata alkaloid at one time by this method. According to the calculation of external standard method, the purity of them were 99.77% and 99.73% respectively determined by UPLC. The structure of the two target product was identified by comparison with standards, ¹H NMR and ¹³C NMR. The structure data of the separated products were in agreement with the literature. The method is suitable for the mass preparation of sanguinarine and chelerythrine, because it has many advantages such as simple post-treatment, low cost, high purity of separation products and strong practicability.