The invention provides azacitidine lyophilized powder for injection. The azacitidine lyophilized powder is prepared from, by weight, 2-8 parts of azacitidine, 2-8 parts of mannitol, 100-300 parts of acetonitrile and 684-896 parts of water. The invention further provides a preparation method of the azacitidine lyophilized powder for injection. The preparation method comprises the steps that part ofthe water for injection is added into a liquid preparation tank; 100-300 parts by weight of acetonitrile is added, and stirring is conducted until the mixture is clear; 2-8 parts by weight of mannitol is added, stirring is conducted until the mixture is completely dissolved, and the temperature is lowered to be 0-4 DEG C; 2-8 parts by weight of a pre-micronized raw azacitidine material is weighed, washed with part of the remaining water for injection and then added into the liquid preparation tank, intensive stirring is conducted until the mixture is dissolved, and the water for injection isadded to reach the constant volume of 1000 parts; after sterile filtration, filling and freeze-drying, the preparation product is obtained. The particle size of the product and the total impurity level of the product can be effectively controlled, the stripping curve and other bioequivalence indexes of the product are equivalent to those of an original research product, and the total impurity level is lower than that of the original research product.