In order to investigate the effects of biological nitrification inhibitors (BNIs) on nitrification and nitrous oxide (N₂O) emission on yellow soil in Southwestern Guizhou, a three-week soil incubation experiment was conducted to explore the effects of two BNIs (methyl 3-(4-hydroxyphenyl) propionate (MHPP) and syringic acid (SA)) on soil inorganic nitrogen content, amoA gene expression of ammonia-oxidizing microorganism and N₂O production. The results show that compared with the control (CK), MHPP and SA can significantly inhibit the nitrification process with the inhibition rates of 6%–43% and 5%–51%, respectively. Moreover, MHPP and SA both reduce the abundance of ammonia-oxidizing archaea (AOA) by 12%–22% and 27%–41%, and the abundance of ammonia-oxidizing bacteria (AOB) by 6%–19% and 26%–46%. Meanwhile, nitrate nitrogen content in yellow soil is significantly positively correlated with AOB abundance during the whole incubation, but has no significant correlation with AOA abundance, indicating that AOB plays a dominant role in the nitrification process. In terms of N₂O emission, MHPP and SA show strong inhibitory effects of 51% and 21% on total N₂O cumulative production, respectively, and the reduction of MHPP is better than SA. MHPP decreases N₂O peak while SA mainly delays the emergence of the peak of N₂O production rate. This study indicates biological nitrification inhibitors MHPP and SA have the potential to reduce nitrogen fertilizer consumption and increase nitrogen use efficiency of the yellow soil in Southwestern Guizhou, which provides a theoretical basis for the development of new green fertilizers for flue-cured tobacco in the future.