Abstract: The aim of this reserach is to research the preparation of highly active atrazine degradation bacteria by using the freeze-drying method, and get the optimum process conditions. Microbacterium sp.HBT4 agents was prepared by using the freeze-drying method, and the process conditions were optimized by the single-factor method and the orthogonal test, with the survival rate of the bacteria as the index. The results showed that the addition of 10% of sucrose (w/v), 8 mL/L of glycerol, and 15% of skim milk (w/v) was the best combination of freeze-drying protectants for the formula. Four influencing factors were selected in orthogonal experiments, and the single-factormethod results determined three levels. The best results were obtained: the centrifugation speed of the bacteria solution was 7000 r/min; the ratio of bacteria to freeze-dried protection agent was 2:1; the amount of liquid in the freeze-drying process was 12 mL; and the time of freeze-drying was 16 hours. This moment, the highest survival rate of freeze-dried bacteria was 75.1%. The results showed that the bacteria prepared under the experimental conditions had a high survival rate and good stability, thus laying a foundation for the production and application of microbial agents.