Chloroform fumigation and denaturing gradient gel electrophoresis(DGGE) were employed to study the influence of different fertilizer treatments with crop residue return on microbial biomass and nitrogen-fixing bacteria community in a gray brown alluvial paddy soil at soil depth of 0-10 cm and 10-20cm. Results show that soil microbial biomass carbon (SMB-C), microbial biomass nitrogen (SMB-N) and diversity of nitrogen-fixing bacteria decreased with soil depths. The control treatment with straw removal had the lowest amounts of SMB-C and SMB-N. Among the treatments receiving straw return, the quantity of SMB-C and SMB-N in the treatments with low nitrogen rate and omitting was significantly lower than the full rate NPK treatment. Omission of phosphate on the basis of NPK nonsignificantly reduced the SMB-N compared to the NPK treatment. It shows that straw mulching with addition of adequate NPK fertilizers can significantly increase the SMB-C and SMB-N. The diversity indices of DGGE profiles indicate that the treatment with adequate NPK had the most abundant nitrogen-fixing bacterial diversity. According to UPGMA cluster analysis, dendrogram of the 10 treatments were different, and the micro-organisms at the 0-10cm and 10-20cm in the control treatment differed from all the other treatments and stood alone in a separate group. DGGE bands sequencing further revealed that the 14 closely related species were largely uncultured bacteria nifH gene fragment with the predominant community falling in the class of Proteobacteria in Betaproteobacteria. Application of PCR-DGGE can well interpret the community structure characteristics of the nitrogen-fixing bacteria molecules in the gray-brown alluvial paddy soil with straw return and different fertilizer treatments.