Abstract: In order to explore the causes of tobacco root rot and provide theoretical basis for its prevention and control from the perspective of microbial ecology, Illumina Miseq high-throughput sequencing was used to analyze the effect of tobacco root rot on the fungus community structure and diversity in rhizosphere soil of tobacco plants. The results showed that the occurrence of root rot had a significant influence on the richness of fungi in tobacco rhizosphere soil. Abundance of OTU in the rhizosphere soil at healthy and root rot infected tobacco plants was 904 and 647, respectively. The total number of OTUs of healthy tobacco was 1.40 times of that of root rot infected tobacco, and its unique OTUs was 2.59 times of that of root rot infected tobacco. Analysis of the composition and diversity of fungi showed that Shannon, ACE, and Chao1 index of the rhizosphere soil at root rot infected tobacco were reduced by 29.81%, 40.12%, and 40.61%, compared with healthy plants; and the fungus diversity level in rhizosphere soil at root rot infected tobacco was significantly lower than that of the healthy tobacco plants. At the same time, the dominant species of fungus community in rhizosphere soil at root rot infected tobacco and healthy tobacco plants were significantly different. The relative abundance of tobacco root rot pathogen (F. solani) increased by 303.45% compared with the healthy tobacco plants, which was 4.03 times of the healthy tobacco. Meanwhile, the relative abundance of Trichoderma asperellum and Penicillium raperi was increased by 17.62% and 50.46%, compared with the healthy tobacco plants. The above results indicate that the change of fungus community structure and the reduction of fungus species diversity in the rhizosphere soil are important characteristics of tobacco root rot occurrence. This study could provide scientific basis for the early prevention or ecological regulation of tobacco root rot.