Abstract：Objective To purify the starting material of etimicin sulfate, gentamicin C1a (HPLC purity 91%~93%) by macroporous adsorption resin chromatography and the salting out crystallization method, thereby reducing the impurity level of etimicin from gentamicin C1a. Methods The adsorption capacity and elution parameters of gentamicin C1a purified by the macroporous adsorption resin were investigated by the adsorption amount of gentamicin C1a and the purity of gentamicin C1a. Based on the purity and the salting out yield of gentamicin C1a sample after salting out, the optimum temperature, solvent, acid addition amount and acid type of salting-out process were investigated. Results The superior resin NM200 was obtained, and the preferred analytical solution had a pH of 2.0 and a flow rate of 0.5BV/h. After optimization, the purification yield increased from 65% to 74%. Through salting-out crystallization conditions screening and optimization, it was determined that the conditions of adding sulfuric acid (pH6.5) and methanol and ethanol 1:2 were better after adding solvent at room temperature. The purity of gentamicin C1a obtained after optimization was 98.2%, and the yield was more than 93%. A variety of inorganic acid precipitation crystals were studied, and it was found that white solids were precipitated under the conditions of phosphoric acid, sulfuric acid and carbonic acid. Conclusion By comparing the purification connection of the macroporous adsorption resin and the salting out crystallization, the purity of gentamicin C1a free base obtained after combination and purification is greater than 99.0%, which is more than 6% higher than the sample before purification, and the yield is more than 70%.