The invention discloses a method for removing hyocholic acid from chenodeoxycholic acid. The method comprises the steps of dissolving, residue removing, complexing, crystallizing, centrifugal separating, emulsifying, acidifying, drying, mother liquor treating and the like; high-boiling-point butyl acetate is utilized as solvent, volatilization of the solvent is reduced, a good separation effect is achieved by utilizing the unique dissolution property of butyl acetate to impurities, and the solvent belongs to a three-level low-toxicity dangerous chemical; the selective complexing characteristic of sec-butylamine to bile acid is utilized, and the hyocholic acid and other impurities can be separated to a higher degree. According to the separation and purification method, impurity removing is qualified in one time, the loss rate of the chenodeoxycholic acid is 5% or below and is greatly lower than the effective ingredient loss of 30% in a conventional crystallization method, the solvent can be recycled, three wastes are not generated, and the method is an environment-friendly, low-cost and efficient separation method.