Abstract: To clarify the mechanisms of biochar and AM fungi on tobacco growth, a field experiment was conducted, and the effects of biochar and AM fungi on the root morphology, physiological characteristics and chemical constituents of flue-cured tobacco were studied. The results showed that the application of biochar, G. intraradices (Gi) and G. mosseae (Gm) significantly increased the volume, area, length and vigor of tobacco roots and improved their morphological structure. The application of biochar had no obvious effect on tobacco photosynthesis, but the application of biochar combined with Gi or Gm significantly increased net photosynthetic rate, stomatal conductance, transpiration rate and water use efficiency, while significantly decreased intercellular CO₂ concentration, which indicated that the photosynthesis of tobacco leaves was promoted. The application of biochar combined with Gi or Gm also improved the harmony of chemical constituents and quality of flue-cured tobacco. Overall, the application of biochar combined with Gi or Gm had positive effects on root morphology, photosynthesis and chemical constituents of tobacco.