Abstract: Intercropping is an important agricultural measure to improve soil ecological effect, mitigates obstacles associated with continuous cropping, and improve crop yield and benefit. This study investigated and analyzed the changes in the rhizosphere soil microbial community structure and physicochemical properties in tobacco (NC102) and soybean (Linbean10) under intercropping and monoculture modes. It also evaluated the effects of these changes on crop agronomic traits and disease incidence. The results showed that the intercropping of tobacco and soybean increased the soil pH, the contents of available phosphorus and exchangeable magnesium. Simultaneously, it significantly altered the composition of rhizosphere microbial community and increased the abundance of beneficial microorganisms such as Proteobacteria and Bacillus. Under the intercropping mode, the plant height of tobacco was increased by 8.0% and the leaf length and leaf width were also higher than those of monoculture. The disease investigation results showed that the disease indices of root and stem diseases and angular leaf spot under intercropping mode decreased by 60% compared to monoculture mode, while the disease index of viral disease was only 25% of that under monoculture mode. This study can provide a scientific basis for promoting the integrated cultivation system of grain, economic, and tobacco crops for achieving stable yields.