A field experiment was conducted to evaluate the combined effects of biochar (B) and Trichoderma-enriched bio-organic fertilizer (O) on soil quality and leaf chemical composition in flue-cured tobacco. Each amendment was applied at three rates: 0 (B₁, O₁), 750 (B₂, O₂), and 1 500 kg/hm² (B₃, O₃). Soil nitrogen and potassium availability, microbial biomass carbon (MBC) and nitrogen (MBN), enzyme activities, and leaf nicotine content were determined. Relative to B₁, B₂ and B₃ raised rosette-stage soil pH by 4.78% and 5.38%, organic matter by 13.41% and 19.11%, total nitrogen by 4.38% and 5.11%, respectively. Across both sampling stages, the two biochar rates increased alkali-hydrolyzable nitrogen by 10.16%-35.39%, available potassium by 3.73%-19.66%, MBC by 3.73%-43.75%, and MBN by 19.23%-67.31%; urease and nitrate reductase activities were enhanced by 6.96%-22.62% and 38.80%-90.47%, respectively. These improvements translated into higher leaf nitrogen (6.20%-11.54%), potassium (22.45%-35.19%), and nicotine (10.46%-80.54%), along with a lower nitrogen-to-nicotine ratio (5.71%-38.13%). For the Trichoderma biofertilizer, O₂ and O₃ relative to O₁ increased maturity-stage alkali-hydrolyzable nitrogen by 11.86% and 13.11%, available potassium by 5.89% and 10.06%, elevated rosette-stage MBC by 5.54% and 7.41%, while suppressing nitrate reductase activity by 8.08% and 9.33%. Consequently, upper-leaf nitrogen, potassium, and nitrogen-to-nicotine ratio increased by 7.41% and 2.53%, 9.24% and 25.77%, 3.09% and 15.97%, respectively, whereas upper-leaf nicotine declined by 4.93% and 10.34%, middle-leaf nitrogen-to-nicotine ratio decreased by 9.84% and 6.70%. Redundancy analysis identified rosette-stage soil pH and maturity-stage MBN as the primary determinants of middle-leaf quality, whereas rosette-stage MBN and maturity-stage total nitrogen governed upper-leaf quality. This pattern revealed a temporal shift in regulatory drivers, from early pH and MBN control to late nitrogen-supply dominance. These findings demonstrated that biochar and Trichoderma biofertilizer act in a complementary manner—biochar retaining nutrients and Trichoderma enhancing their bioavailability—to synergistically improve both soil fertility and leaf chemical quality in rainy tobacco-growing regions.