Soil microbial community reflecting soil quality affects the soil ecosystem functions and has been suggested as the sensitive indicator for predicting change of soil ecosystems. To understand the influence of organic manure and chemical fertilizers on the microbial biomass and community in soils of the North China Plain, soil samples were collected from the 0-20 cm tilled layer in a long-term experimental field at the Fengqiu state key agroecological experimental station, Fengqiu County, Henan Province, China, and used to measure the phospholipid fatty acid profiles of microorganisms. The study included three treatments with four replicates: control (CK, without fertilization), organic manure (OM) and chemical fertilizer NPK (NPK). The long-term application of fertilizer NPK and particularly organic manure significantly increased the concentrations of soil organic C, total N, available P and available K, and reduced soil pH compared to treatment CK. This finding indicates that organic manure application or balanced fertilization of N, P and K could efficiently improve the soil fertility, and the growing conditions were more suitable for crops and soil microorganisms. The long-term application of organic manure significantly increased the amount of total microbial PLFAs by 15.4% compared to the value in the CK treatment, but the addition of fertilizer NPK had no significant effect. Manuring significantly increased the amount of bacterial PLFAs from 41.03 nmol/g soil in the CK treatment to 48.03 nmol/g soil in the OM treatment, but did not change the amount of fungal PLFAs, which resulted in the significant reduction of the ratio of fungi to bacteria. Fertilization slightly, but not significantly, increased the amount of bacterial PLFAs and reduced the amount of fungal PLFAs, resulting in the slight decrease of the ratio of fungi to bacteria. However, manuring and fertilization dramatically reduced the magnitude of actinomycetal PLFAs, indicating that organic manure and chemical fertilizers both inhibited the growth of actinomycete in arable soils.After the 21-year (1989-2009) application of organic manure and chemical fertilizers, only 52% of total PLFAs showed similarity among the three treatments. Principle component analysis (PCA) of PLFA was used to describe changes in microbial community,and the result showed that change in microbial community among three treatments was mainly driven by the application of fertilizers and could be attributed to the first and second principal components, which accounted for 43.1% and 30.9% of the total variation, respectively. Three treatments were located in different quadrant: the OM treatment distributed in the fourth quadrant, whereas the NPK treatment in the third quadrant and the CK treatment in the second quadrant. In the OM treatment, bacteria containing a19 ∶ 0, br14 ∶ 0, 16 ∶ 1ω5c and 17 ∶ 1ω9, and fungi containing 18 ∶ 1ω10c were dominant microorganisms, while bacteria with 18 ∶ 1ω7, i19 ∶ 0, br18 ∶ 0, 16 ∶ 1ω7t and a15 ∶ 0 for treatment NPK. In contrast, no dominant microorganisms could be identified in the CK treatment.