Abstract: 【Objective】The study was to explore the rational application amount of bio-organic fertilizer for its large scale application and extension in tobacco in Guangdong.【Method】The bio-organic fertilizers were fermented with isolated beneficial microbial strains (Azotobacter sp. X34 ; Bacillus sp. jb21 and Rubrivivax gelatinosus sbg11) and organic matter. The effects of different amounts of bio-organic fertilizers on tobacco growth, leaf photosynthetic pigment content, leaf enzyme activity, and the yield and output value of flue-cured tobacco were studied through field tests.【Result】Under field conditions, different application amounts of bacterial fertilizers could significantly improve tobacco plant height, stem girth, leaf length and leaf area, promote tobacco photosynthetic pigment content and metabolic enzyme activity, and increase the proportion of superior tobacco leaf and yield and output value of tobacco leaf. T3 treatment(the application amounts forbase fertilizer and top dressing were 20 g per plant respectively) significantly promoted the growth of tobacco, with the highest increase in plant height (44.53%). The yield and output value of T3 treatment increased by 29.67% and 18.87%, respectively. 【Conclusion】The application amount of bacterial fertilizer in T3 (in which 20g bacterial fertilizers were applied for base fertilizer and top dressing per plant respectively) can increase the yield and quality of tobacco obviously, and can be used as the recommended dosage of fertilizer.