Abstract: To clarify the physiological effects of combined application of selenium (Se) and molybdenum (Mo) on tobacco's resistance to black shank, K326 was used to establish a combined application system. The effects of combined Se and Mo application on tobacco disease incidence, photosynthetic characteristics, cellular structure, antioxidant metabolism and hormone content (mass fraction) were analyzed through the inoculation of Phytophthora nicotianae. The results showed that the combined Se and Mo treatment significantly reduced the disease incidence and the black shank index, with superior effects than those of the single-element treatments. This combination also raised chlorophyll content, optimized photosynthetic parameters, and repaired chloroplast disintegration and the cell wall structural damage caused by Phytophthora nicotianae infection. By activating the actions of antioxidases catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and defense enzymes nitrate reductase (NR), polyphenol oxidase (PPO) in the plants, the productions of superoxide anion (O₂^-·) and hydrogen peroxide (H₂O₂) were regulated, inhibiting membrane lipid peroxidation reactions triggered by oxidative stress, thereby reducing the accumulation of lipid peroxidation end products such as malondialdehyde (MDA) in the plants. In the meantime, the contents of auxin (IAA), abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), soluble proteins and soluble sugars in the plants were increased, enhancing the plant's disease resistance. Therefore, the combined application of Se and Mo regulates the contents of osmotic adjustment substances by enhancing tobacco photosynthetic, antioxidant, and hormone metabolic capabilities, thereby improving tobacco disease resistance.