The invention discloses a preparation method for bacillus subtilis freeze-dried powder. The method comprises the following steps: (1) uniformly dispersing bacillus subtilis suspension in a freeze-drying protective agent solution for forming a bacillus subtilis suspension solution, split-charging in a penicillin bottle, pre-cooling in ice-water bath and then putting into liquid nitrogen for pre-freezing; and (2) transferring the pre-frozen bacillus subtilis suspension solution into a freeze dryer and performing vacuum freeze drying, thereby acquiring the bacillus subtilis freeze-dried powder. According to the invention, the bacillus subtilis is adaptively quick vitrified pre-frozen in the manner of pre-freezing and then putting into the liquid nitrogen, so that the thallus loss in the pre-freezing process is reduced, the vacuum drying parameter is optimized, the frozen damage and dry stress damage are effectively relieved, the rejuvenation survival rate of the bacillus subtilis freeze-dried powder is greatly increased and the acquired bacillus subtilis freeze-dried powder has property stability, is convenient for long-term storage and long-distance transportation under room temperature and is suitable for scientific research or being used as a biopesticide.