The invention discloses a calf spleen extract injection prepared by reversed-phase column chromatography and a preparation method thereof. The original calf spleen extract solution is purified by reversed-phase column chromatography with simple process, so as to remarkably improve the stability of product and the quality standard of pharmacologically active substance. The inventive method utilizes the reversed-phase column chromatography instead of conventional adsorption, elution and ion-exchange column chromatography, so as to reduce production process and meet the requirement of improving product quality standard. Compared with the prior calf spleen extract solution, the inventive product has the advantages of low toxic and side effects and low content of pigment impurities. The No.7 peak area in HPLC chromatogram is improved from 17.66% before reversed-phase chromatography to 51.82% after reversed-phase chromatography, thus indicating high unit content of effective component.