In order to clarify the rational application method of biochar-based fertilizer in tobacco agriculture and reveal its regulatory effect on the rhizosphere soil microecology of flue-cured tobacco, 'Yunyan 87' in Wushan tobacco area of Chongqing was used as the test variety in this study. Through the field randomized block experiment, the biochar-based fertilizer application group T and the non-fertilizer control group C were set up, with three replicates. Combined with 16S rRNA gene high-throughput sequencing technology, the effects of biochar-based fertilizer (600 kg / hm²) on the bacterial community in the rhizosphere soil of flue-cured tobacco were analyzed. The results showed that after the application of biochar-based fertilizer, the number of bacterial OTUs in the rhizosphere soil of flue-cured tobacco increased, and the richness and diversity levels of soil bacteria were enhanced. Compared with the control, at the phylum level, the abundance of Chloroflexi and Acidobacteria increased after the application of biochar-based fertilizer. At the genus level, species abundance of Pseudomonas, Streptomyces, Rhodanobacter, Acidothermus and Actinospica in rhizosphere soil of flue-cured tobacco treated with biochar-based fertilizer were significantly different. Among them, the abundance of Pseudomonas and Acidothermus showed an increasing trend. Cluster analysis showed that significant differences in bacterial community composition between the treatment and control groups. A total of 82 significantly different bacterial species were found through LEfSe (LDA Effect Size) analysis. Among them, the abundance of 10 significantly different species increased after treatment with biochar-based fertilizer, such as Xanthobacteraceae and Intrasporangium, and most of them belong to beneficial and resistant bacterial categories. Therefore, the application of biochar-based fertilizer altered the bacterial community structure in the rhizosphere soil of flue-cured tobacco and improved the beneficial bacterial species in the rhizosphere soil. Future research may focus on the screening and identification of differential species, which will provide technical support for the rational use of biochar-based fertilizer to improve soil microbial environments in tobacco agricultural production.