This research investigated the N2O fluxes and community compositions of nitrifiers and denitrifiers in a winter wheat-summer maize rotation system on the North China Plain. The experiment included three treatments: 1) a control treatment (CK); 2) biochar at 10.0 t ha–1 yr−1 (C); and 3) organic fertilizer at 7.5 t ha−1 yr−1 (M). The application of biochar reduced the cumulative N2O emissions by 47.7% and 62.2% in the maize and wheat seasons, respectively, compared with those of the CK treatment. Organic fertilizer increased the cumulative N2O emissions by 311.1% in the maize season and had no significant effects on them in the wheat season. Organic fertilizer reduced the cumulative N2O emissions by 39.8% in the nonfertilizer period of the maize season. The cumulative N2O emissions in the maize season accounted for 75.2–90.0% of the annual emissions among all the treatments. Biochar and organic fertilizer affected soil N2O emissions mainly by changing soil denitrifiers. In the maize season, the lower abundances of Candidatus Nitrosoarchaeum (AOA-amoA), Nitrosomonas (AOB-amoA), Mesorhizobium (nirK), Magnetospirillum (nirS) and Halomonas (nirS) may result in lower N2O emissions in the biochar treatment, and organic fertilizer had a similar influencing mechanism on N2O emissions during the nonbasic fertilizer and nontopdressing periods of the maize season. In the wheat season, the dominant genus Alicycliphilus (nosZ) was the major contributor to decreasing N2O emissions in the biochar treatment, while there was no biomarker related to N2O emissions in the M treatment. The soil pH, NO3−-N content and water-filled pore space (WFPS) were the key factors shifting the community compositions of nitrifiers and denitrifiers in this study. The application of biochar could be a better practice to improve saline-alkali soil with lower N2O emissions. These findings will improve our understanding of the nitrifiers and denitrifiers response to biochar and organic fertilizer.