Nitrification is believed to be one of the primary processes of N2O emission in the agroecological system, which is controlled by soil microbes and mainly regulated by soil pH, oxygen content and NH4+ availability. Previous studies have proved that the relative contributions of ammonia oxidizing bacteria (AOB) and archaea (AOA) to N2O production were varied with soil pH, however, there is still no consensus on the regulating mechanism of nitrification-derived N2O production by soil pH. In this study, 1-octyne (a selective inhibitor of AOB) and acetylene (an inhibitor of AOB and AOA) were used in a microcosm incubation experiment to differentiate the relative contribution of AOA and AOB to N2O emissions in a neutral (pH = 6.75) and an alkaline (pH = 8.35) soils. We found that the amendment of ammonium (NH4+) observably stimulated the production of both AOA and AOB-related N2O and increased the ammonia monooxygenase (AMO) gene abundances of AOA and AOB in the two test soils. Among which, AOB dominated the process of ammonia oxidation in the alkaline soil, contributing 70.8% of N2O production derived from nitrification. By contrast, the contribution of AOA and AOB accounted for about one-third of nitrification-related N2O in acidic soil, respectively. The results indicated that pH was a key factor to change abundance and activity of AOA and AOB, which led to the differentiation of derivation of N2O production in purple soils. We speculate that both NH4+ content and soil pH mediated specialization of ammonia-oxidizing microorganisms together; and both specialization results and N2O yield led to the different N2O emission characteristics in purple soils. These results may help inform the development of N2O reduction strategies in the future.