The invention discloses a preparation method of a B component reference substance in lincomycin hydrochloride. The preparation method comprises the following steps: dissolving lincomycin hydrochlorideof which the content of B component is greater than or equal to 3.0 percent into water and absorbing with a resin column; washing and removing impurities; adsorbing the lincomycin hydrochloride solution of which the content of the B component is smaller than or equal to 0.7 percent by using the resin column; washing and collecting washing water after the adsorption is finished; carrying out second-time resin separation and purification, pre-washing with a solvent, removing impurities and then desorbing with the solvent and collecting a desorption solution; concentrating the collected desorption solution and discharging to obtain lincomycin hydrochloride B component wet powder; carrying out centrifugal treatment on the lincomycin hydrochloride B component wet powder and then pulping for 0.5 hour; drying the lincomycin hydrochloride B component wet powder under vacuum conditions to obtain a lincomycin hydrochloride B component reference substance with the purity of 98 percent or above.The resin separation and purification method disclosed by the invention for preparing lincomycin hydrochloride B component reference substance has the advantages of low preparation cost, simple operation and easiness in realization.