Abstract: Spray drying is a commonly used method in the food industry since it is cost-effective and highly flexible, but spray drying typically results in lower survival rate of starter cultures than freeze-drying. In this study, the influence of heat pre-treatment (46 ℃, 1 h) and oxidative pre-treatment (0.5 mmol/L H₂O₂, 1 h) on the spray dried powder of Lactobacillus rhamnosus hsryfm 1301 was investigated. The results showed that heat pre-treatment and oxidative pre-treatment could improve the survival rate. The survival rate of the untreated group was only 29%, while the survival rates of heat pre-treated and oxidative pre-treated cells were 98% and 76%, which increased by 3.38 and 2.62 folds, respectively. At the same time, the acid-resistant of the untreated bacterial powder was 0.8%, while the acid-resistant survival rates of heat pre-treated and oxidative pre-treated bacterial powder were 76% and 72%, which increased by 95.0 and 92.5 times, respectively. Moreover, the heat pre-treated and oxidative pre-treated bacterial powder had good storage ability in the environment of −20 ℃. The survival rate of heat pre-treated bacterial powder remained greater than 80% after stored at −20 ℃ for 4 months, and that of oxidative pre-treated bacterial powder was greater than 40%. It was suggested that the strengthened heat stress and oxidative stress tolerance of L. rhamnosus hsryfm 1301 by heat pre-treatment and oxidative pre-treatment were helpful to survive spray drying.