In the large cross stage of tobacco seedlings, the roots of tobacco seedlings were treated with Brevibacillus laterosporus by dipping root(T1), irrigation root(T2) and immersion root + irrigation root(T3), and the root viability, soil microbial species and quantities, soil enzyme activity, tobacco leaf chemical quality and economic traits were determined 30, 60 and 90 days after transplanting, and the results showed that 30 and 60 days after transplanting, the root activity and soil enzyme activity of tobacco treated with T1, T2 and T3 were significantly increased compared with CK(without B. laterosporus). Compared with CK, the root viability of tobacco in T3 was significantly increased by 39.49%, the number of bacteria and actinomycetes in the soil of tobacco rhizosphere increased by 40.51% and 41.64%, respectively, and the number of fungi decreased by 28.39%. The conventional chemical composition of the middle leaves of tobacco with T1, T2 and T3 all reached the standard of high-quality tobacco leaves, and the proportion and output value of middle and upper tobacco increased by 19.51% and 72.57% respectively compared with CK, and the chemical composition of the middle leaves was more coordinated than that of other treatments.