Effects of different nitrogen application methods on microbial community structure of paddy soil at different rice growth stages were examined using phospholipid fatty acid analysis (PLFA) and Biolog technique. There were four treatments, no straw returning or fertilization (CK), straw returning +urea with the proportions of after wheat harvest, before rice transplanting, tillering stage and booting stage being 0:6:2:2 (T) and 3:3:2:2 (T), and straw returning+co-application of biogas slurry and urea with the proportion of after wheat harvest, before rice transplanting, tillering stage and booting stage being 3 (biogas slurry):3 (2biogas slurry+1urea):2 (urea):2 (urea)(T). Results showed that T significantly increased soil available nitrogen contents at all growth stages, which was significantly higher at maturity stage than that at tillering and booting stages. T-T had higher available phosphorus and available potassium contents at all growth stages compared with CK, which were higher at tillering stage than at booting and maturity stages. The interaction between growth stage and treatment in paddy soil significantly affected the contents of soil available nitrogen, available phosphorus and available potassium. Furthermore, carbohydrate, amino acid, polymer and carboxylic acid were the primary carbon sources for microbial community of paddy soil. T effectively enhanced soil carbon sources metabolic utilization intensity. The interaction between growth stage and treatment in paddy soil significantly affected the microbial utilization capacity of carbohydrates and carboxylic acids. Soil microbial biomass was significantly higher in T and T treatments. Moreover, T had high fungi/bacteria (F/B) value, indicating that fungi could benefit the stabilization of paddy soil. In summary, simultaneous nitrogen application (urea or biogas slurry) and straw returning could increase soil microbial activity and improve soil environment in paddy field.