Ammonia oxidation driven by ammonia-oxidizing archaea (AOA) and bacteria (AOB) plays a significant role in the nitrogen cycle, but the mechanism of this important action in reservoir sediments is unclear. In this study, based on quantitative PCR and Illumina high-throughput sequencing of the ammonia monooxygenase (AMO) gene, the abundance, diversity and community structure of AOA and AOB in Miyun Reservoir sediments were investigated. The key environmental factors for regulating community structure, as well as the response of AOA and AOB abundance and transcriptional activity to external ammonia and organic carbon, were also explored. The results showed that the diversity and abundance of AOB in the reservoir sediments were higher than those of AOA, and the community structure of AOA showed a different spatial distribution characteristics compared with those of AOB. AOA community structure was closely related to ammonia nitrogen concentration and water depth, while that of AOB were not significantly affected by environmental factors. AOB were more sensitive to concentration variations of ammonia nitrogen and organic carbon from exogenous sources. Therefore, in Miyun Reservoir sediments, AOA were more sensitive to the influence of environmental factors in the reservoir area, while AOB were more sensitive to the influence of exogenous sources.