【Objective】Mixed culture technology of soil suspension and culture medium, high-throughput sequencing, and conventional analysis methods were used to study the influence of long-term fertilization on inorganic phosphorus (P) mobilization in soil. 【Method】Soil samples were collected from the plots of a 15-year-long experiment regarding crop cultivation and fertilization in Zunyi City, China. The treatments included (i) fallow soil (Control), (ii) consecutive flue-cured tobacco cultivation plus the application of only chemical fertilizers (C-CF), (iii) consecutive flue-cured tobacco cultivation plus the application of chemical fertilizers in combination with manures (C-CFM), (iv) flue-cured tobacco-maize rotation plus the application of only chemical fertilizers (R-CF), and (v) flue-cured tobacco-maize rotation plus the application of chemical fertilizers in combination with manures (R-CFM). The soil collected from the long-term fertilization test site was divided into two parts. A part of the soil sample was used for the mixed culture technology of soil suspension and culture solution. The method involved preparing the soil suspension (soil: sterile water = 1: 100) and then diluting it to 10^-5 with sterile water. 200 mL diluent was taken and inoculated into NBRIP (National Biological Research Institute phosphate growth medium) solid medium with Ca₃ (PO₄)₂, FePO₄ and AlPO₄ as phosphorus sources. After incubation at 30 ℃ for 2 days, the number of colonies with a phosphorus dissolving circle was observed. Then, another 200 mL 1: 100 soil suspension was taken and inoculated in 50 mL NBRIP liquid medium with Ca₃ (PO₄)₂, FePO₄ and AlPO₄ as phosphorus sources for 16 days. At 12, 24, 48, 72, 96, 120 and 144 hours after inoculation, the culture medium was drawn, and the pH, soluble inorganic phosphorus content, and organic acid content were determined. The other part of soil samples was sent to Meggie Biological Company for high-throughput sequencing to study the effect of long-term fertilization on the relative abundance of enzyme synthesis pathway related to organic acid synthesis. 【Result】The number of cultivable microorganisms (bacteria, fungi, actinomycetes and inorganic phosphate bacteria) in flue-cured tobacco-maize rotation, application of organic fertilizer and Control soil increased, and the population diversity increased. When the soil suspension was inoculated in a microbial culture medium, the content of available phosphorus was higher in the flue-cured tobacco-maize rotation than in consecutive flue-cured tobacco cultivation. Also, the available phosphorus was higher in the application of chemical fertilizers in combination with manures and Control than when only chemical fertilizers were applied; among which R-CFM was the highest and C-CF was the lowest. In the mixture, hydrogen ions were significantly correlated with dissolved Ca₃ (PO₄)₂, and organic acids were significantly correlated with dissolved FePO₄ and AlPO₄. This indicates that compared with pure culture technology of phosphorus-mobilizing microbes, soil suspension culture can not only reflect the phosphorus-mobilizing characteristics of microorganisms but also be closer to the complex phosphorus solubilization status of soil microbial flora. In high-throughput sequencing, the relative abundance of the synthetic pathways of citrate (Si) -synthase, protein glucose dehydrogenase and malate synthase in the soil of R-CFM was significantly higher than that of other treatments. 【Conclusion】 Optimal crop cultivation and scientific fertilization are conducive to the activation of soil inorganic phosphorus, and R-CFM can be promoted as a flue-cured tobacco demonstration cultivation model for widespread promotion in Zunyi tobacco areas. Soil suspension culture can be used as an extension of pure microbial liquid culture as (a) it is closer to the complex phosphorus solubilization status of soil microbial flora, (b) effectively characterizes the ability of PMM to activate inorganic phosphorus, and (c) supplements the deficiencies of pure microbial liquid culture technology. The difference in the relative abundance of enzyme synthesis pathways related to the synthesis of organic acids also explains the reason for rational crop rotation and combined application of chemical fertilizers and manures to promote the activation of soil inorganic phosphorus from the perspective of molecular biology.