The invention relates to a decitabine freeze-dried powder injection and a preparing method thereof. The prepared decitabine freeze-dried powder injection is used for treating myelodysplastic syndrome (MDS). The decitabine freeze-dried powder injection contains decitabine, utilizes the mixed solvent composed of the tert-butyl alcohol and the injection water in the preparation process, wherein the concentration of the decitabine in the mixed solvents is 2.5-5 mg / ml; and the volume ratio of the solvents is: 5-50% of tert-butyl alcohol and the balance of injection water. The potassium dihydrogen phosphate and the sodium hydroxide may be added for the pH regulator. The preparation process comprises the following steps: measuring tert-butyl alcohol, adding injection water, potassium dihydrogen phosphate and sodium hydroxide, stirring and mixing evenly, cooling to 2-15 DEG C, heat preserving, adding decitabine, stirring to dissolve, filtering, filling, plugging, disking, freeze-drying, pressing plug, out box, tying and packing after quality test qualification.