Ammonia-oxidizing archaea (AOA) and Ammonia-oxidizing bacteria (AOB) are key microorganisms in the soil nitrogen cycle, but how they change in the intercropping system, affected by interspecific interaction and N application levels, is not clear. A field experiment of soybean/maize intercropping with three nitrogen application levels was designed. Illumina MiSeq sequencing was used to determine AOA and AOB diversity and communities in the rhizosphere of intercropped soybean and maize. Nitrogen absorption of maize grain has increased by 21.09% to 33.54% in intercropping compared with monoculture, while that of soybean was reduced, especially in 240 kg N<middle dot>ha(-1)(N2). Our results showed that the alpha-diversity of AOA and AOB in the rhizosphere of maize was reduced in intercropping treatment across all N application levels. The opposite results were found in intercropped soybeans. Additionally, there was an increase in the alpha-diversity of AOB in the soybean rhizosphere with N2 treatments. Specifically, alpha-diversity of AOB in intercropped soybean in 240 kg N<middle dot>ha(-1)(N2) increased by 10.45% and 1.6% relative to the 0 kg N<middle dot>ha(-1)(N0) and 180 kg N<middle dot>ha(-1)(N1), respectively. This effect is further magnified within the monocropped maize under 240 kg N<middle dot>ha(-1)(N2), reflecting enhancements of 10.68% and 5.37%, respectively. Under intercropping conditions, the abundance of the dominant AOA genus, Nitrososphaera, significantly decreased more than sixfold under 180 kg N<middle dot>ha(-1)(N1). Conversely, the abundance of the dominant AOB genus, Nitrosospira, increases with the higher nitrogen application rates, although intercropping exerts a diminishing influence. While its trend within the rhizosphere of soybean is the opposite. Moreover, Redundancy Analysis (RDA) and Mantel tests showed a correlation between variations in ammonia-oxidizing microbial communities and soil-available nitrogen content (p = 0.001, r > 0.4). Due to species competition after intercropping, the soil available nitrogen content decreased, resulting in changes in the soil ammonia-oxidizing microbial community. The results indicated that interspecific competition in intercropping systems could change the diversity and composition of AOA and AOB in the rhizosphere of crops, consequently influencing N transformation and enhancing nitrogen uptake. These findings elucidated the mechanisms of how intercropping systems bolster nitrogen-use efficiency through the dynamics of rhizosphere microorganisms.