Quaternary Red Clay (QRC) is the most common planting soil with low soil fertility and low crop yields in Southeast China, with low soil fertility and low crop yields. Many factors can impact the fertility and utilization efficiency of QRC. Here, we conducted a long-term fertilization experiment from 1984 to 2013. Five fertilization measures were carried out, including non-fertilization group; chemical Fertilizer group; 70% chemical and 30% organic fertilizer group; 50% chemical and 50% organic fertilizer group; 30% chemical and 70% organic fertilizer group. Soil organic matter (OM), total nitrogen (TN), total phosphorus (TP), total potassium (TK), soil microbial biomass carbon (SMBC) and nitrogen (SMBN), and soil enzymes activity were measured to evaluate the changes of soil. In addition, soil microorganisms were determined by high-throughput sequencing technology, and the dominant microbes were screened. The higher the proportion of organic fertilizer was, the higher the soil OM content was. The OM content of the non-fertilization group was the lowest. Similarly, SMBC and SMBN showed a consistent trend with OM content. Illumina sequence results showed that the application of organic fertilizer reduced the relative abundance of Chloroflexi, Acidobacteria and Nitrospirae, but increased Proteobacteria and Actinobacteria. The relative abundance of Acremonium and Mortierella were also greatly increased by different fertilization strategies. However, when high proportion of organic fertilizer was applied, the abundance of Acremonium and Mortierella decreased. Long-term balanced inorganic fertilization (NPK, 60%N:20%P:20%K) can effectively improve the quality and fertility of QRC. The effect of different fertilization strategies on fungi was greater than that on bacteria. The change of soil microorganism also proved the validity of inorganic fertilizer application.