Abstract: In order to explore the effects of organic-inorganic biological fertilizer and chemical fertilizers on the yield, quality, and soil properties of flue-cured tobacco, a field comparative experiment was conducted with different application rates of organic-inorganic biological fertilizer (0, 180, 300 and 600 kg/hm²) and four treatments of chemical fertilizer combination. The effects of organic-inorganic biological fertilizer and chemical fertilizer combination on soil hardness, microbial quantity, photosynthetic parameters, nitrogen and potassium accumulation, and chemical composition of tobacco leaves after curing were explored. The results showed that compared with CK, the application of organic-inorganic biological fertilizers and chemical fertilizer could reduce soil surface hardness and subsurface hardness, increase net photosynthetic rate of tobacco leaves, increase tobacco yield by 6.5%~11.8%, and significantly increase the number of soil bacteria, fungi, and actinomycetes during the clumping stage by 4.2%~63.7%, 14.7%~62.1% and 9.9%~62.8%, respectively. After baking, the total sugar, reducing sugar, total nitrogen, total potassium, and nicotine content of tobacco leaves significantly increased by1.4~4.3, 2.6~4.0, 0.2~0.7, 0.1~0.3 and 0.3~0.9 percentage points, respectively. In summary, the application of organic-inorganic biological fertilizer and chemical fertilizers can improve soil hardness, increase the number of soil microorganisms, enhance the net photosynthetic rate of tobacco, promote tobacco growth, increase tobacco yield, and optimize the chemical composition of tobacco. Among them, the application of chemical fertilizer+organic-inorganic biological fertilizer (300 kg/hm²) has the best effect, and it is recommended to promote its application in the tobacco-growing areas of southern Henan.