Due to the extensive use of fertilization and herbicides, farmland ecosystems in high-yield areas are areas where productivity is declining, biodiversity is reduced, and soils are seriously degraded. Soil microbes as soil organisms participate in soil carbon, nitrogen, and phosphorus and sulfur cycles and play an important role in soil fertility. In addition, microbial community composition can be used to assess soil fertility and health. Based on phospholipid fatty acid analysis, the effects of different carbon to nitrogen ratio fertilizers on soil microbial community structure were studied by pot experiment. The results showed that organic fertilizers and organic-inorganic combined fertilizers with low C/N ratios could increase the content of soil microbial biomass carbon and nitrogen (SMBC and SMBN) in soil, soil nitrogen has high biological activity and high nitrogen utilization. So the wheat biomass of the applications of straw, pig manure combined with chemical fertilizer (C/N ratio 12.5) and straw combined with chemical fertilizer (C/N ratio 12.5) are much significantly higher than those of the organic-inorganic combined fertilizers (C/N ratio 40) application; When the carbon to nitrogen ratio of organic fertilizer is 10:1 and 5:1, the SMBC: SMBN is lower, and soil nitrogen has higher biological activity and higher nitrogen utilization rate. When the carbon to nitrogen ratio is high (20:1 and 15:1), the SMBC: SMBN is higher, and soil nitrogen activity is lower. This study provides a theoretical basis and practical basis for the application of organic fertilizer in organic agricultural science.