Abstract:[Objective] Study on the technology of selection and optimization of Bacillus natto freeze-dried powder, in order to increase the survival rates of living bacteria. [Methods] Single factor and orthogonal experimental designs were taken to measure the survival cells and then screened the protection agent formulations. And the storage stabilities of optimized lyoprotectant powder prepared at protection agent formulations. And the storage stabilities of optimized lyoprotectant powder prepared at ?20 °C, 4 °C and 25 °C were tested. [Results] The effective protecting agents of Bacillus natto were: skimmed milk powder, mannitol, L-sodium ascorbate. The best protectants formula: 10% skim milk powder, 4% Mannitol and 1% L-sodium ascorbate, the results showed that the survival rate could reach to 91.63%. The survival rates of fungus powder conserved at ?20 °C, 4 °C, 25 °C for 12 months were 88.79%, 70.16% and 10.52%, respectively. The storage stability of bacteria powder at ?20 °C and 4 °C were excellent, while that at 25 °C was poor. [Conclusion] The freeze-dried powder protective of Bacillus natto was optimized, which provided a guidance for application of Bacillus natto, stabilization of quality of live bacterial product and development of novel live bacterial products.