Abstract: To investigate the application effects of carbon nanosol (CNS) in tobacco-planting soils of southwestern Guizhou, field experiments were conducted in Qianxiang Subdistrict of Anlong County, Yuzhang Town of Xingren City, and Zhuchangping Town of Xingyi City.This study examined the impacts of supplementary applications of carbon nanosol at different rates (T1: 200 mL/plant; T2: 400 mL/plant) on soil fertility based on conventional fertilization. Ten indicators, including soil pH, 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 experimental site variations. The results showed that carbon nanosol application reduced soil pH and affected soil nutrient contents and microbial properties across 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 increases exceeding 45%, while showing relatively smaller improvements in alkali-hydrolyzable nitrogen and urease activity. The T2 treatment (400 mL/plant) significantly increased urease activity by an average of 60.5%, with other indicators increasing by 4.7% to 31.9%. PCA indicated that both application rate and experimental site jointly drove 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 serve as an effective regulatory measure for improving soil fertility in tobacco-planting areas of southwestern Guizhou, with 200 mL/plant identified as a more suitable application rate that significantly enhances soil nutrient contents and microbial activity.