Novel preservation condition without ultra-low temperature is needed for the study of pathogen in marine fishes. Freeze-drying is such a method usually used for preservation of terrigenous bacteria. However, studies using freeze-drying method to preserving marine microorganisms remain very limited. In this study, we optimized the composition of protectants during the freeze-drying of Edwardsiella tarda, a fish pathogen that causes systemic infection in marine fishes. We found that the optimal composition of protectant mixture contained trehalose(8.0%), skim milk(12.0%), sodium citrate(2.0%), serum(12.0%) and PVP(2.0%). Orthogonal and interaction analyses demonstrated the interaction between serum and skim milk or sodium citrate. The highest survival rate of E. tarda was observed when the concentration of Na Cl was 10.0, 30.0 and between 5.0 and 10.0 g L~(-1) for preparing TSB medium, E. tarda suspension and protectant mixture, respectively. When E. tarda was frozen at-80℃ or-40℃ for 6 h, its survival rate was higher than that under other tested conditions. Under the optimized conditions, when the protectant mixture was used during freeze-drying process, the survival rate(79.63%–82.30%) of E. tarda was significantly higher than that obtained using single protectant. Scanning electron microscopy(SEM) image indicated that E. tarda was embedded in thick matrix with detectable aggregation. In sum, the protectant mixture may be used as a novel cryoprotective additive for E. tarda. Novel preservation conditions without ultra-low temperature is needed for the study of pathogen in marine fishes. However, studies using freeze-drying method to preserving marine microorganisms remain very limited. We found that the optimal composition of protectant mixture contained trehalose (8.0%), skim milk (12.0 Orthogonal and interaction analyzes demonstrated interaction between serum and skim milk or sodium citrate. The highest survival rate of E. tarda was observed when the (%), sodium citrate (2.0%), serum (12.0%) and PVP concentration of NaCl was 10.0, 30.0 and between 5.0 and 10.0 g L -1 for preparing TSB medium, E. tarda suspension and protectant mixture, respectively. When E. tarda was frozen at -80 ° C or-40 ° C fo Under the optimized conditions, when the protectant mixture was used during freeze-drying process, the survival rate was (79.63% -82.30%) of E. tarda was significantly higher than that obtained using single protectant. Scanning electron microscopy (SEM) image indicated that E. tarda was embedded in a thick matrix with detectable aggregation. In sum, the protectant mixture may be used as a novel cryoprotective additive for E. tarda.