【Objective】Nitrifiers play an important role in the process of farmland soil nitrogen transformation. A study on the distribution of nitrifying microorganisms in aggregates will help to reveal the interaction mechanism between soil structure, microorganisms and soil nutrient cycling.【Method】The changes in soil aggregate properties and nitrification potential (N_P) in maize monoculture (M-M) and maize peanut rotation (M-P) were compared, and the abundance and community composition of different nitrifying functional genes in soil aggregates were evaluated by quantitative PCR and high-throughput sequencing.【Result】Compared with M-M, M-P significantly increased pH, NH₄⁺ and total carbon (TC) in soil aggregates. M-P also significantly enhanced N_P, but the aggregate size had no significant effect on N_P. The abundance of the AOB amoA gene was higher in M-P and also more abundant in smaller aggregates. The distribution pattern of AOA and comammox amoA genes was opposite to that of AOB, indicating that AOB can better adapt to the micro-environment of smaller aggregates, while AOA and comammox tended to dominate in larger aggregates. In addition, compared with M-M, the difference in AOA/AOB and comammox/AOB ratio between M-P aggregates decreased, indicating that rotation promoted an even distribution of soil nitrifiers among different aggregate sizes. By further analysis of the nitrifying community based on the 16S rRNA gene sequencing, the results showed M-P increased the proportion of Nitrolancea-like NOB and Candidatus Nitrosocosmicus-like AOA, decreased the proportion of Nitrospira-like NOB, and had no significant effect on AOB compositions. The aggregate size only had a significant effect on the proportion of Nitrosospira-like AOB. NH₄⁺ content and pH were the main factors affecting soil N_P and the nitrifying microbial community structure among soil aggregates. N_P was positively correlated with AOB amoA gene abundance and negatively correlated with AOA amoA gene abundance. However, in terms of community composition, Nitrosospira-like AOB, Candidatus Nitrosocosmicus-like AOA and Nitrospira-like NOB all showed positive correlations with N_P.【Conclusion】Soil aggregate size and cropping system can greatly affect the distribution of nitrifying microorganisms in soil aggregates. However, nitrifying microorganisms have different adaptation mechanisms among aggregates. This study provides a theoretical support for improving the ecological adaptation mechanism of soil nitrifying microorganisms in the micro-environments under Gramineae-Legume rotation.