Abstract: 【Objectives】 Soil microbes play key functions in organic matter decomposition, nutrient cycling and regulation of plant growth. Investigating the effect of long-term chemical fertilizers application on microbial characteristics is critical for the improvement in rational fertilizer application, and provides theoretical understanding in sustainability of agricultural management. 【Methods】 The experiment was based on the long-term experiment in National Monitoring Base of Soil Fertility and Fertilizer Efficiency on Loess Soil in Yangling City, Shaanxi Province. Six treatments were chosen, including no fertilization control (CK), nitrogen fertilizer only (N), nitrogen and potassium fertilizer(NK), phosphorous and potassium fertilizer(PK), nitrogen and phosphorous fertilizer(NP), and nitrogen, phosphorous and potassium fertilizer(NPK). Biolog Eco-plate was applied to test the effect of long-term chemical fertilization on soil microbial characteristics. Moreover, soil microbial biomass and soil microbial community functional diversity were determined. 【Results】 Compared to the control (CK), the soil microbial biomass C (SMBC) and soil microbial biomass N (SMBN) did not change in N treatment. However, the microbial quotient (qMB) and the N utilization (SMBN/TN) were reduced significantly under N treatment. SMBC, SMBN concentration and qMB were increased in NP and NPK treatments, where the shift in soil microbial community structure was also found. But N utilization was not increased in these treatments. In NK and PK treatments, SMBC, SMBN concentration and qMB were slightly increased, while soil microbial community structure did not show much difference. Biolog analysis showed that soil microbial activity was increased under the treatments including P (PK, NP and NPK). At the initial stage of incubation, the metabolism activity was greater under the treatments with P than those without P (CK, N and NK). Nitrogen application alone did not affect microbial community functional diversity, while other fertilization treatments improved the ability of carbon source utilization as indicated by substrate richness, Shannon richness index, and dominant index (Simpson index) of soil microbial community with the increase greatest in NP and NPK treatments. The evenness index of soil microbial community was not affected by different chemical fertilizer applications. Principal component analysis showed that the ability for carbon substrates utilization varied with the greatest difference caused by the application of inorganic N and P fertilizers. 【Conclusions】 Balanced chemical fertilization (NP and NPK) largely improves soil microbial diversity under winter wheat-summer maize rotation in the anthropogenic loess soil area.