The invention discloses a separation and purification method of atisine chloride atractydin monomers. The method includes: using an ethanol solution to perform heating extraction on rhizoma atractylodis for 2-3 times, combining filtrate and concentrating until no alcohol exists; B, extracting the concentrated solution with ethyl acetate for 2-4 times, and concentrating the ethyl acetate; C, adding silica gel into the ethyl acetate, and performing sample mixing and drying; D, loading the mixed sample into a mounted silica gel column for silica gel column chromatography, eluting with pure petroleum ether, and using a thin-layer chromatography point plate to confirm and collect production sections; E, concentrating the collected eluted production sections, freezing to separate out crystals, and using a Buchner funnel to fast filter the atisine chloride atractydin crystals while the crystals are cold, wherein HPLC detection shows that the purity of the crystals can reach above 99%, and the crystals can be used as the comparison products of content measuring. The separation and purification method has the advantages that the method is simple in process, short in production cycle, easy in cost control and suitable for industrial production, organic reagents can be recycled, and the obtained atisine chloride atractydin monomers are high in purity.