【Objective】 The heavy use of pesticides such as chlorothalonil (CTN) in agricultural production inevitably has an impact on the soil's ecological environment, thus, exacerbating nitrous oxide (N₂O) emission from farmland soils, considered an important emission source of greenhouse gasses. However, there are few studies on how CTN affects agricultural soil N₂O emission and its associated microbial mechanism. 【Method】 In this study, the response characteristics of N₂O emission in three kinds of farmland soils (S1-vegetable field soil, S2-rice field soil, and S3-wheat field soil) to different concentrations of CTN were studied through indoor culture experiments. The concentration gradients of chlorothalonil were set as 0(CK), 5(T5), 10(T10) and 25 mg·kg^–1(T25). The abundance of microbial functional genes was analyzed simultaneously through real-time quantitative PCR. 【Result】 The results showed that (1) Compared with CK, the treatments of T5, T10, and T25 in the acidic vegetable field soil of S1 led to an increase of 1 868%, 1 264%, and 232% in N₂O emissions, respectively. In the S2 neutral paddy soil, T5, T10, and T25 respectively led to an increase of 4%, 138%, and 334% in N₂O emissions, while for the alkaline wheat field soil S3, T5, T10, and T25 led to an increase of 230%, 119%, and 23% in N₂O emissions respectively. (2) The addition of CTN changed soil physicochemical and microbial characteristics, and significantly increased soil dissolved organic carbon (DOC) content, ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), nitrite-reducing bacteria (nirK, nirS), N₂O reducing bacteria (nosZ), and total bacteria (16S rRNA) genes abundance. Correlation analysis showed that the increase in N₂O emissions was mainly related to the increase in soil carbon and nitrogen substrate contents whereas the abundance of microbial functional genes was mainly attributed to the increase in the gene abundance of total bacteria, ammonia-oxidizing bacteria, and nir-type denitrifying bacteria, as well as the increase in soil DOC content. 【Conclusion】 CTN can increase soil DOC content and microbial gene abundance, and eventually lead to an increase in soil N₂O emission. Also, the effect of CTN on soil N₂O emission depends on soil properties and application concentration. The results of this study provide a scientific basis for further understanding of soil N₂O emission driven by CTN application and its microbiological mechanism and also have important significance for understanding the potential ecological and environmental risks of agricultural CTN application.