The invention discloses a preparation method of a quantitative microorganism freeze-dried product. The preparation method comprises the following steps of: diluting microorganisms at a stable period to obtain uniform diluent and preparing the diluent into bacterial suspension with quantitative concentration; mixing the bacterial suspension with a protecting agent to obtain mixed liquor; and bottling quantitative mixed liquor, pre-freezing at the temperature of -30 DEG C, freezing and drying in vacuum, and sealing in vacuum to obtain the quantitative microorganism freeze-dried product, wherein the protecting agent comprises the following components in parts by weight: 0.1-10 parts of water-soluble sugar, 0.1-5 parts of defatted milk powder, 0.1-5 parts of antioxidant, 0.1-20 parts of gelatin and 0-10 parts of active carbon. The microorganism freeze-dried product prepared by adopting the preparation method contains basically quantitative microorganisms, and is convenient to use; and the activity of the microorganisms can be ensured when the product is stored at the temperature of below 8 DEG C, and the biological and chemical properties of the microorganisms do not change basically within the retention period.