Abstract: Ammonia oxidation, driven by the ammonia oxidizers, is the rate-limiting step of nitrification and plays a key role in soil nitrogen cycling. In this study, real-time PCR and Illumina MiSeq sequencing approaches were used to investigate the effects of long-term different fertilization regimes on the abundance and community structure of ammonia oxidizers, targeting the amoA genes, in a 30-year located experimental paddy soil in Ningxiang County, Hunan Province. Four treatments were compared, including control without fertilizer (CK), fertilizers NPK (CF), 70% NPK plus 30% manure (CFM₁), and 40% NPK plus 60% manure (CFM₂). The results showed that the abundance of amoA genes in AOA and AOB was in the range of 3.09×10⁷-8.37×10⁷ and 1.04×10⁷-7.03×10⁷ copies·g^-1 dry soil, respectively. Fertilization significantly increased the AOA and AOB abundances. However, no significant difference was observed in AOB abundance between CFM₂ and CK. Manure fertilization rate greatly affected the α diversity index of AOB rather than AOA. The Shannon index of AOA and ACE and Chao1 indexes of AOB observed in CFM₁ were significantly higher than that in CK, respectively. Thaumarchaeota and Crenarchaeota were the predominant AOA phyla and Nitrosospira, environmental_samples_norank, Bacteria_unclassified and Nitrosomonadales_unclassified were the main AOB genus groups which accounted for 83.4% and 97.8% of the total AOA and AOB amoA gene reads, respectively. Venn diagram indicated that manure fertilization rate had a stronger effect on the OTU number of AOB amoA gene than that of AOA in different treatments, but it slightly altered the proportion of shared AOA and AOB amoA gene reads. Furthermore, there were pronounced differences in the community structure of AOB among different treatments than that of AOA. These results suggested that manure fertilization rate significantly affected the abundance, diversity and community structure of AOA and AOB. The Shannon index of AOA and the abundance and ACE and Chao1 indexes of AOB in CFM₁ were significantly higher than that in the rest treatments, respectively. Our results provided basis for further exploring the response mechanism of ammonia oxidizers to different fertilization strategies and the roles of ammonia oxidizers in nitrogen transformation in agricultural systems.