In this study, low vacuum and room temperature plasma (LVRTP) was employed to pretreat Bacillus amyloliquefaciens CICC 10035. The optimal process conditions were screened by changing parameters including discharge power, treatment time and working pressure. The differential proteins were analyzed by bioinformatics using proteomics technology, and the effects of plasma pretreatment on cell growth and α-amylase secretion were discussed. The results showed that the optimal LVRTP pretreatment conditions were as follows: discharge power 120 W, treatment time 15 s, working pressure 135 Pa. Under the optimal conditions, α-amylase activity in the fermentation was more than 400 U/ml after 48 h, about 20% higher than the control. Proteomic analysis exhibited that LVRTP pretreatment had strong positive effects on protein synthesis and cell metabolism, especially in DNA repair and amino acid synthesis. On the one hand, LVRTP pretreatment improved the activity of cells and delayed the aging process; on the other hand, it promoted the synthesis and secretion of α-amylase in cells.