The linkage between N2O emissions and the abundance of nitrifier and denitrifier genes is unclear in the intensively managed calcareous fluvo-aquic soils of the North China Plain. We investigated the abundance of bacterial amoA for nitrification and narG, nirS, nirK, and nosZ for denitrification by in situ soil sampling to determine how the abundance of these genes changes instantly during N fertilization events and is related to high N2O emission peaks. We also investigated how long-term incorporated straw and/or manure affect(s) the abundance of these genes based on a seven-year field experiment. The overall results demonstrate that the long-term application of urea-based fertilizer and/or manure significantly enhanced the number of bacterial amoA gene copies leading to high N2O emission peaks after N fertilizer applications. These peaks contributed greatly to the annual N2O emissions in the crop rotation. A significant correlation between annual N2O emissions and narG, nirS, and nirK gene numbers indicates that the abundance of these genes is related to N2O emission under conditions for denitrification, thus partly contributing to the annual N2O emissions. These findings will help to draw up appropriate measures for mitigation of N2O emissions in this 'hotspot' region.