Soil N plays a critical role in plant nutrition, which is controlled by nitrification. Biochar can enhance the N concentration of soil, which is thought to affect nitrification, especially when combined with chemical fertilizer (biochar fertilizer). However, there are several reports on biochar fertilizers that affect nitrifying communities. The related mechanism is also not understood. In this study, the nitrifying community analysis results suggested that the abundances of ammonia-oxidizing archaea in treatments BT3 and BT5 and ammonia-oxidizing bacteria (AOB) in treatment BT4 were significantly higher than those in other biochar fertilizer treatments, whereas nitrite-oxidizing bacteria (NOB) in treatment BT2 exhibited a more significant difference (p < 0.05). Compared with that in BT1, the AOB Nitrosomonas (51.74%) and NOB Nitrolancea (62.26%) became the dominant bacteria (p < 0.05) as the biochar fertilizer treatment increased. The metabolic composting and molecular docking theory precisely and simply illustrated that 2,2-diethylacetamide, which is similar to the oxalate ion, interacts with the active center of pyruvate kinase, which affects the glycolysis pathway. A potting experiment proved that the microbial composition originated from 2,2-diethylacetamide out of 17 biochar extracts, which increased pyruvate kinase gene expression from 1.37 to 11.03 (p < 0.05), pyruvic acid of pot soil from 1.73 to 21.65 (p < 0.05), and nitrifier abundance from 0.20% to 0.56% (p < 0.05). The soil Cu (R2 = 0.21; p < 0.05), Mo (R2 = 0.53; p < 0.05), Zn (R2 = 0.37; p < 0.05), and TOC (R2 = 0.24; p < 0.05) contents were negatively correlated with nitrifier abundance as the biochar fertilizer increased. Our study illuminates the mechanism of the effect of biochar and soil elements on the nitrifier community.