Abstract: In order to analyze the relationship between rhizosphere soil microecological factors and the occurrence of tobacco root-knot nematode disease, a typical tobacco field with tobacco root-knot nematode disease was selected in Aziying Township, Songming County, Kunming City, Yunnan Province. Rhizosphere soil samples were collected from both diseased and healthy tobacco plants. The number of second-instar larvae of root-knot nematodes, soil chemical properties, enzyme activities, and microbial community structure were analyzed and compared. The results showed that the root-knot nematode pathogen was identified as Meloidogyne incognita. Compared with the rhizosphere soil of healthy tobacco plants, the number of root-knot nematodes in the rhizosphere soil of diseased tobacco plants increased by 4.29 times (p<0.01). Soil acid phosphatase activity, pH, and available phosphorus content were significantly reduced by 86.98 % (p <0.05), 0.33 units (p<0.01), 45.53 % (p<0.05), respectively. In contrast, sucrase activity was significantly increased by 57.36 % (p<0.05). The microbial balance, bacterial diversity and richness decreased, and the fungal diversity increased but the richness decreased. The bacterial genus Sphingomonas were enriched in the rhizosphere soil of the diseased tobacco plants, and the fungal phylum Ascomycota were enriched in the rhizosphere of the healthy tobacco plants. The number of 2nd instar larvae of root-knot nematode was significantly positively correlated with sucrase activity and Sphingomonas, and significantly negatively correlated with pH, acid phosphatase, available phosphorus and Ascomycota. Low soil pH, acid phosphatase activity and available phosphorus content, and high sucrase activity may be important factors in inducing tobacco root-knot nematode disease.