Abstract: To address the problems of excessive application of chemical fertilizers and the instability of tobacco yield and quality in the Hengyang tobacco-growing area, the effects of partially substituting chemical fertilizer with organic fertilizer combined with plant growth-promoting rhizobacteria (PGPR) on root development, nutrient distribution, leaf yield and quality formation of flue-cured tobacco were investigated. Under equal nutrient input, treatments included conventional fertilization (CK), substitution of chemical nitrogen fertilizer with organic fertilizer at different proportions (20%, 30%, and 40%), and combined application of PGPR inoculant. Agronomic traits of tobacco plants, root characteristics, nutrient contents in leaves and roots, chemical components of cured tobacco leave, and economic traits were measured at key growth stages. The results showed that the treatment with organic fertilizer replacing 20% of nitrogen (N) fertilizer combined with PGPR inoculant (CBM-20%) significantly enhanced root activity at all growth stages, optimized root architecture (with the greatest total length, surface area, volume, and average diameter), and increased N, phosphorus, and potassium contents in both leaves and roots, particularly enhancing root potassium uptake. Compared with CK, the CBM-20% treatment resulted in more balanced potassium-to-chlorine ratio and sugar-to-nicotine ratio in tobacco leaves, and increased total N content, total potassium content, yield, output value, and the proportion of middle- and upper-grade tobacco leaves by 28.9%, 78.1%, 60.5%, 60.5%, and 36.1%, respectively. Correlation analysis indicated that the chemical components and economic benefits of cured tobacco leaves were all significantly positively correlated with root activity, root architecture (root length, surface area, and root volume), plant height, and maximum leaf length. In conclusion, in the Hengyang tobacco-growing area, using organic fertilizer to replace 20% of chemical N fertilizer combined with PGPR inoculant can effectively enhance tobacco yield and quality by optimizing root architecture, improving root activity, and strengthening nutrient uptake, thereby achieving the synergistic goals of reducing chemical fertilizer inputs, increasing nutrient use efficiency, and promoting green production of flue-cured tobacco.