The invention discloses a preparation method of high-purity hyodeoxycholic acid. The preparation method comprises the steps: weighing raw materials, adding strong alkaline and water, saponifying at the temperature of 95-100 DEG C for 16-24h, cooling to room temperature, standing, siphoning to remove a supernatant liquid, adding water into paste in the lower layer, stirring for dissolving the paste, adding strong acid to acidize until a congo red test paper turns to be blue, adding ethyl acetate, stirring for extracting for 20-50min, standing for layering, removing a water phase, adding water into the ethyl acetate to wash until the pH value of the water phase is equal to 6-7, adding the ethyl acetate into anhydrous sodium sulfate to dehydrate, carrying out activated carbon decoloration, filtering, concentrating until precipitates are separated out, cooling, filtering or squeezing and drying in vacuum; adding an alcohol solvent, stirring for dissolving the above product, slowly adding an alkaline organic solvent, stirring, cooling, separating out a great number of precipitates, filtering and drying; and adding water and sodium hydroxide, stirring for dissolving, dropwise adding hydrochloric acid with a volume concentration of 1:1, stirring, filtering and drying to obtain the hyodeoxycholic acid. The invention aims to provide the preparation method of the hyodeoxycholic acid with simple process, high yield and high purity.