This study focused on tobacco plant, which is highly sensitive to N supply, and utilized various tobacco-growing soils from across the country. Through incubation experiments, pot and field experiments, a soil testing method for N fertilization in tobacco fields was optimized. The results showed that under aerobic conditions, the increase in soil ammonium nitrogen was significantly negatively correlated with pH, while nitrate nitrogen was significantly positively correlated with pH. The increase in total inorganic nitrogen was primarily positively correlated with multiple soil N availability indicators. Both increasing temperature and extending incubation time favored the accumulation of ammonium nitrogen under anaerobic conditions. When accounting for initial nitrate nitrogen lost during anaerobic incubation, the anaerobic and aerobic methods generally reflected consistent soil N supply capacities. Inorganic nitrogenobtained under specific incubation conditions more accurately reflected the N supply capacity of different soils compared to single common N indicators. Using stepwise regression, a formula for tobacco leaf N uptake based on alkali-hydrolyzable nitrogen and inorganic nitrogen was derived: Tobacco leaf N uptake (mg/kg soil)=37+0.3×alkali-hydrolyzable nitrogen+0.7×inorganic nitrogen. This formula outperformed all other single soil N indicators. By comparing with the appropriate field N application rates, a simplified formula for soil testing-based N fertilization in tobacco fields was proposed: Recommended N application rate for tobacco fields (kg/hm²)=135-1.07×soil baseline N supply indicator, where soil baseline N supply indicator=0.3×alkali-hydrolyzable nitrogen+0.7×inorganic nitrogen. This formula is expected to provide an effective methodological basis for future soil testing-based N fertilization in tobacco fields.