In this study, lincomycin was successfully purified by macroporous adsorption resin column chromatography using the HZ3 resin. The optimal separation parameters were set as follows: the column bed height was 33 cm, sample loading capacity was 48 mg/mL and flow rate of loading was 1 mL/min. A mixture of 0.02 mol/L of Na 2 HPO 4 ∙12H 2 O (pH = 8.5, adjusted using H 3 PO 4 ) and acetone (80:20, v/v) was used as the eluent. The elution flow rate was maintained at 3 mL/min. Under these parameters, the purity of lincomycin calculated using the standard curve was 99.00 %, with the yield being 97.84 %. This enrichment and separation method of lincomycin is highly regarded owing to its remarkable efficiency and straightforward operation. Thus, the proposed method for the separation and purification of lincomycin holds considerable promise for pharmaceutical applications.