The invention discloses a preparation method of an ulinastatin freeze-dried powder preparation, belonging to the field of pharmaceutical preparations. According a prescription for the ulinastatin freeze-dried powder preparation, every 1000 bottles of the ulinastatin freeze-dried powder preparation comprise 25,000,000 to 100,000,000 units of ulinastatin, 0-20 g of mannitol, 0-2 g of sodium chloride, 0-2 g of disodium hydrogen phosphate and 0-2 g of sodium dihydrogen phosphate. The preparation method for the ulinastatin freeze-dried powder preparation provided by the invention has the followingcharacteristics: preparation is implemented at room temperature, so the problem that industrial production temperature is low and difficult to control is overcome, and energy consumption is lowered; and a phosphate buffer solution is prepared at room temperature at first, and then the active ingredient ulinastatin is added, so the buffer solution has protective effect on the active ingredient ulinastatin. The ulinastatin freeze-dried powder preparation prepared by using the prescription and the preparation method provided by the invention is convenient for industrial production, reduces energyconsumption and can improve the stability of the active ingredient--an ulinastatin solution.