Abstract: To investigate the effects of carbon nanosol application in tobacco-planting soils of southwestern Guizhou Prefecture, field trials were conducted in Qianxiang Subdistrict of Anlong County, Yuzhang Town of Xingren City and Zhuchangping Town of Xingyi City. The effects of supplementary applications of carbon nanosol at different rates (T1: 200 mL/plant; T2: 400 mL/plant) on soil fertility based on conventional fertilization was examined. Ten indicators including soil pH value, alkali-hydrolyzable nitrogen, available phosphorus, available potassium, urease activity, sucrase activity, microbial biomass nitrogen, microbial biomass carbon, respiration rate, and respiratory quotient were measured and analyzed. Principal component analysis (PCA) and Mantel test were employed to explore the influence mechanisms of application rate effects and variations among experimental sites. The results showed that carbon nanosol application reduced soil pH value and affected soil nutrient contents and microbial properties across the different experimental sites. Compared with the control, the T1 treatment (200 mL/plant) demonstrated the most significant enhancement effects on available phosphorus, sucrase activity, microbial biomass carbon, microbial biomass nitrogen and soil respiration rate, with average increasing rates all exceeding 45%, while the increasing rates of alkali-hydrolyzable nitrogen and urease activity were comparatively smaller. The T2 treatment (400 mL/plant) significantly increased urease activity by an average of 60.5% and other indicators by 4.7%-31.9%. Principal component analysis showed that both application rate and experimental site resulted in the variations in soil indicators. Mantel test further revealed that soil respiration rate was more sensitive to carbon nanosol application rate. In conclusion, carbon nanosol can be regulated to effectively improve soil fertility in tobacco-planting areas in southwestern Guizhou, with 200 mL/plant as a proper application rate that significantly enhances soil nutrient contents and microbial activity.