Abstract: To investigate the conversion and supply mechanism of soil nutrients in tobacco field, determine scientific fertilization procedure and construct healthy soil environment, a field experiment was conducted with three soil treatments: without fertilizers (CK), with chemical fertilizer (CF), with organic manure and chemical fertilizer combination (MCF). Soil enzyme activities and bacterial community structures in the rhizosphere of flue-cured tobacco were studied. The results showed that comparing with CK, there was an increment of microbial carbon and nitrogen and a stimulation of enzymatic activities including dehydrogenase, invertase and urease in the soil of MCF treatment. The numbers of OTUs of 16S rDNA in the soils of MCF, CF and CK treatments were 291, 245 and 208, respectively; which belonged to 17 bacterial phyla, such as Proteobacteria, Actinobacteria, Firmicutes, etc. There were a lot of Nitrobacter and Nitrosospira. Among 20 dominant bacteria, the abundance of Cellulomonas, Fibrobacter, Coprococcus and Enterococcus was higher in the soil of MCF treatment than that of CK treatment, which was beneficial to organic matter mineralization and organic fertilizer utilization. Comparing with CK treatment, the abundance of Desulfobacter and Nocrdioides increased, and that of Acinetobacter decreased, while Rhizobium disappeared in the rhizosphere soil of CF treatment. The uniformity index and diversity index of bacterial community in the soil of MCF treatment was significantly promoted, the dominance index significantly reduced. In conclusion, MCF treatment improved soil environment and increased bacterial biodiversity, which were helpful to inhibiting the over reproduction of pathogenic bacteria and decreasing the disease risk of tobacco plants.