Ammonia oxidation is a rate-limiting step of nitrification and an important index to evaluate nitrogen cycling in soil. Real-time PCR, terminal restriction fragment length polymorphism and clone library analysis were used to investigate the effects of long-term different fertilization regimes on the abundance and community structure of ammonia oxidizers in a long-term localization experiment in the dry farming station of the Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences. Five treatments were compared including chemical nitrogen (N) fertilization, chemical nitrogen, phosphorous and Potassium fertilization (NPK), manure only (O), NPK plus manure (NPK+O), and no fertilizer as control. The results showed that the abundance of ammonia-oxidizing bacteria (AOB) and nitrification potential were significantly increased in N, NPK, and NPK+O treatments. The sequences of AOB in the control soil were mostly affiliated with genus Nitrosospira clusters 3a.1. The soil treated with N increased the abundance of AOB that belonged to Nitrosospira clusters 3a.2. In contrast, the O-treated soil showed more abundant AOB that belonged to Nitrosospira clusters 3b. In the semi-arid ecosystem, the soil pH and soil moisture were identified as the dominant drivers of the AOB community structure. The abundance of ammonia-oxidizing archaea (AOA) was only increased by applying of urea, while different fertilizations did not have significant effect on the AOA community structure.