Abstract: It is of great significance to increase the mineralization rate of organic fertilizer through oxygen addition measures to meet the fertilizer demand law of flue-cured tobacco growth and development. In this study, the most conducive oxygen addition method including two types of physical oxygenation and three types of chemical oxygenation for soil activity and tobacco plant growth was selected, and the optimal oxygen concentration and time were determined on this basis. Field experiments with different oxygen addition methods were carried out, and the effects were evaluated by field yield, yield value, organic fertilizer mineralization rate and soil microbial community structure. Our results showed that: (1) based on the indicators of tobacco plant growth, agronomic traits and soil ecological diversity, it was found that calcium peroxide had the best effects in chemical oxygen addition while pump oxygenation was best in physical oxygen addition; (2) The 12 h/d oxygenation time and 40 g/plant calcium peroxide of manure were the best, and the root dry weight increased by 1.92 times and 1.24 times compared with the control, respectively; (3) The output value with field application of organic fertilizer combined with calcium peroxide increased by 10.47%, 12.48% and 18.82% in Zunyi, Bijie and Qianxinan respectively. Tobacco quality in calcium peroxide oxygenation treatments increased by 13.97, 3.38 and 19.95 points respectively, and the Industrial utilization increased by 4.5 percentage points compared with the control; (4) Compared with control, the mineralization rate of nitrogen with perforated oxygenation increased by 31.6%~57.1%, while that with calcium peroxide increased by 95.0%~138.9%. In conclusion, perforated oxygenation and calcium peroxide addition can be applied to field production, which improves carbon and nitrogen conversion efficiency, soil microbial functional diversity as well as tobacco yield and quality, thus laying a foundation for the upgrading application of organic fertilizer in flue-cured tobacco.