A field experiment was conducted with different application rates of biochar (B) and Trichoderma bio-organic fertilizer (O): 0 kg/hm2 (B1 or O1), 750 kg/hm2 (B2 or O2), and 1500 kg/hm2 (B3 or O3). The contents of soil nutrients such as carbon (C), nitrogen (N) and potassium (K), microbial biomasses, enzyme activities, as well as the contents of total N (TN), total K (TK), nicotine and N/nicotine ratio in middle and upper leaf were determined. Results showed that compared with B1 plots, B2 and B3 plots not only increased the soil pH, organic matter and TN contents by 2.97%-18.9%, the contents of available N and K by 3.73%-49.8%, the contents of microbial biomasses of C and N (MBC, MBN), microbial quotient for C and N, enzyme activities, and the geometric means of soil enzyme activity (GMea) and total enzyme activity index (Et) by 3.73%-90.5%, but also significantly increased the contents of TN, TK and nicotine by 6.20%-80.5%, while decreasing the N/nicotine ratios by 5.71%-38.1% in middle and upper leaves, respectively. Compared with O1 plots, O2 and O3 plots increased the contents of soil available N and available K at rosette stage by 3.21%-13.1%, as well as the contents of MBC, MBN, urease activity, Et and GMea by 5.53%-43.1%, while increased the TN and TK contents by 2.53%-25.8% and decreased nicotine content by 4.93% and 10.3% in upper leaf, then improving its ratio of N/nicotine by 3.09% and 6.70%, respectively. In addition, the RDA analysis implied that the soil pH, MBN content at rosette stage and the MBN, TN contents at mature stage were dominant factors, which affecting the chemical quality of middle and upper leaf, respectively. Hence, combining biochar and Trichoderma bio-organic fertilizer can synergistically improve soil health and tobacco leaf quality in rainy tobacco-growing areas.