Abstract: For the preparation of Directed Vat Set ( DVS) of Lactobacillus acidophilus NX2- 6, the effect of different protective agents on the survival rate of Lactobacillus acidophilus NX2- 6 during the vacuum freeze- drying was investigated.Influences of centrifugation conditions on the survival rate of Lactobacillus acidophilus NX2-6 was first examined.Then single- factor experiments and orthogonal experiments were carried out to obtain the optimum protective agents formula according to the survival rate of Lactobacillus acidophilus NX2-6 after vacuum freezedrying. The results showed that most optimum centrifugation conditions for preparing the suspension were8000 r / min and 10min ( at 4℃) . The optimal protective agent formula for Lactobacillus acidophilus NX2-6 were as following: malt extract powder 15%, sodium glutamate 3% and inulin 7%. Using this protective agent formula, the survival rate of Lactobacillus acidophilus NX2-6 after vacuum freeze- drying was up to 82.43%.