In order to explore the effective protective substances for freeze-drying Mycoplasma bovis （M. bovis）， this study employed a four-factor， four-level orthogonal design experiment alongside a single-factor experiment. The effects of skim milk， sucrose， mannitol， glucan， trehalose， urea， BSA， lysine， and other substances on the survival rate of M. bovis were detected by measuring the number of the live M. bovis HB150 post freeze-drying and the optimal concentrations and ratios of the different protective agents were ultimately established. The ratios of the main and auxiliary components of the freeze-drying protectants were determined through orthogonal design experiments， and the final ratios of the lyophilized protectants were as follows： 15% skimmed milk， 4.0% sucrose， 4.0% mannitol， 3.0% glucan， 2.0% alginate， 1.5% urea， 1.0% BSA， and 0.3% lysine. When freeze-drying at -40 ℃ and 12 Pa for 20 h， this formulation maintained the highest activity rate of M. bovis， reaching （55.2±2.1）%. The results indicated that the selected dynamic dry protectants exhibited a strong protective effect and could be effectively applied in the actual freeze-drying process of M. bovis.