The invention discloses an injection of calf blood de-protein extract and preparation method, wherein 1ml of the injection comprises solid 41-60mg, 55-65wt% of inorganics, 35-45wt% of organic active compound. The making process comprises the following steps, (1) stirring carf blood or serum, charging sodium citrate into the plasma, homogenizing and storing by refrigeration, separating and collecting plasma for later use, (2) passing the plasma through pearl form chitosan chromatography column, charging ethanol, stirring and centrifuging the protein precipitate, reclaiming ethanol from the supernatant fluid, charging proteinases, enzymolyzing, (3) charging distilled water into enzymolysis liquid, heating and filtering with chitosan modified activated charcoal column, (4) passing the filter liquor through modified activated charcoal column, passing through microporous filtration film under pressurization, loading, sealing, finally carrying out hot-pressed sterilization.