Numerous studies have reported the effect of biochar addition on the structure of soil microbiota, but little information is available on the response of microbial composition to biochar application in soil aggregates. In this study, a single amendment of biochar was applied to a typical paddy field in southern China. Following two years, soil samples separated by a wet sieving method, and the microbial community structure was analyzed using Illumina MiSeq Next Generation sequencing. Results showed that the application of biochar (1) increased significantly the stability of soil aggregates and the proportion of the macro-aggregates fraction (2000-250 μm), and (2) changed significantly the relative abundances of bacteria and fungi among soil aggregates. For instance, the relative abundances of Proteobacteria, Ascomycota, Betaproteobacteriales, and Pleosporales decreased with the increase of aggregate size in C0 treatment, while the relative abundances of Acidobacteria, Basidiomycota, Myxococcales, and Paraglomerales increased gradually. In addition, the application of biochar (3) altered significantly the composition of microbes in different soil aggregates. Compared with C0 treatment, the relative abundances of Proteobacteria, Mortierellomycota, and Betaproteobacteriales increased significantly by 14.37%, 33.28%, and 33.82%, respectively, in the macro-aggregates fraction (2000-250 μm) under biochar amendment, while the relative abundances of Acidobacteria, Ascomycota, and Myxococcales decreased significantly by 20.15%, 19.93%, and 17.66%, respectively, in the micro-aggregates fraction (250-53 μm) under biochar amendment. Lastly, the relative abundances of Basidiomycota increased significantly by 90.25%, whereas Ascomycota and Mortierellomycota decreased significantly by 12.15% and 12.58%, respectively, in the silt-clay fraction (<53 μm). Overall, the application of biochar in a typical paddy field not only changed the structure and distribution of soil aggregates but also the composition of the soil microbial community in this study.