A preparation method of lyophilized Lactobacillus acidophilus powder. The technological process includes fermentation and cultivation of Lactobacillus acidophilus, separation of fermentation liquid, emulsification of bacterial sludge and vacuum freeze-drying of emulsified liquid. It is characterized by fermentation and cultivation of strains: Lactobacillus strains are connected to a triangular flask liquid medium for 30-40° Cultivate for 10-30 hours at C; then connect to a large fermenter for fermentation at 35-40°C for 10-30 hours; Sterilize at 115*C for 15 minutes; there are also strains that are fermented and expanded for 10-30 hours, fermentation broth The pH value is 5.5~6.5. 0D600 is above 2.0: Emulsion vacuum freeze-drying: first pre-freeze at -60~-40°C for 1~5 hours, then vacuum freeze-dry at -60°C and 1~8Pa vacuum 10~30 hours. The 1.0x10"cfu/g Lactobacillus vulgaris Donggan bacterial powder prepared by the method of the present invention can be stored at -18*C for 18 months, and its activity and stability are not affected.