The invention discloses a preparation method of a composite fat-soluble vitamin freeze-dried powder injection. The preparation method comprises the following steps of: firstly, respectively adding fat-soluble vitamins A, E, K and D2 to polysorbate-80 to form two groups of micelle, and dissolving and mixing the two groups of micelle; secondly, adding water for injection, mixing with a mannitol aqueous solution, adding active carbon for adsorption, and regulating pH value; finally, filtering, bottling, freeze-drying, cover-pressing and sealing to obtain the finished freeze-dried powder injection product, wherein each 3.5 ml of freeze-dried powder injection solution contain 2970-3960 IU of vitamin A, 9-11 IU of vitamin E, 1135-180 ug of vitamin K, 2180-240 IU of vitamin D2, 50 mg of polysorbate-80, 120-180 mg of mannitol and the balance of water for injection. According to the invention, the composite fat-soluble vitamin freeze-dried powder injection and the preparation method thereof have the advantages of little auxiliary materials, simple preparation process, low production cost, safe and reliable quality and convenience for storage and transportation and the like.