The invention discloses photobacterium phosphoreum freeze-dried powder and a preparation method thereof. The photobacterium phosphoreum freeze-dried powder comprises the following components in percentage by weight: 25-35 percent of skimmed milk, 20-24 percent of mycose, 0.7-1.0 percent of arginine, 1-1.5 percent of sodium chloride and the balance of thalli. The photobacterium phosphoreum freeze-dried powder adopts a compound cryoprotectant; and experiments prove that the compound cryoprotectant is greatly improved in freezing resistance, has a higher protective property than a simplex protective agent and has good stability. The average value of luminous intensity of the photobacterium phosphoreum freeze-dried powder provided by the invention is about 1,843mv and approximate to a model expected value 1,820. When the toxicity of ZnSO4.7H2O, K2Cr2O7 and C6H5OH is detected with photobacterium phosphoreum freeze-dried powder prepared in different batches, the results show that the EC50 value variation coefficients detected by the photobacterium phosphoreum freeze-dried powder prepared in different batches are less than 6%.