The invention relates to a preparation method of calcitriol which is a compound material, belonging to the technical field of preparation of drug synthesis and organic compound synthesis. In the invention, the calcifediol is taken as raw material, a majority of produced impurities in the chemical reaction process are removed through medium-pressure liquid phase chromatography, wherein calcifediol carried out sulfonylation and ring closing on toluene, and the medium-pressure liquid phase chromatography is used for removing the impurities; products carry out oxidation reaction, medium-pressure chromatographic column separates and removes the impurities, ring opening is carried out, medium-pressure liquid phase is added for coarsely separating stereoisomeride, and at last the products separated by high-pressure liquid phase chromatography is hydrolyzed and refined to obtain the calcitriol. The invention is reasonably selected, comprehensively applies the advantages of the medium and high pressure liquid phase chromatography, has simple detaching method, can effectively remove the impurities, thoroughly separates the stereoisomeride, has high purity of the calcitriol obtained from separation, has a productive rate of 40 percent, high yield and low cost, shortens production period, is suitable for industrial scale, has application prospect, and provides a time-saving, economic and efficient method for the separation and the preparation of chiral compounds.