Abstract: The field experiments were conducted to study the impacts of tobacco stalk-derived biochar at different levels on soil nutrients, microbial diversity and abundance of flora. The results showed that gradient application of tobacco biochar increased pH of acidic soil, promoted soil organic matter and total N accumulation gradually, and improved the releasing of available K. The results also indicated that the amount of rhizosphere soil microbes increased after the application of biochar, and the greatest increment (3.25 times) was achieved by the group at the level of 80 t/hm². The operational taxonomic unit (OTU) increased by 26.4%. The OTU analysis on bacteria at 97% similarity demonstrated that there was no difference at the top 10 dominant phyla between the groups at different levels. The highest distribution was observed in Proteobacteria that accounted for 47.19% to 54.32% of the total bacteria. This association was also applied to the increase of plant growth-promoting microorganisms (PGPMs). The applications of tobacco biochar can not only improve the soil fertility, but also ease the environmental pollution problems caused by unreasonable use of tobacco stems. However some limitations also existed in the popularization and application of tobacco stalk-derived biochar in tobacco agriculture.