To explore the response of soil acidification，ammonia-oxidizing bacteria(AOB)，and ammonia-oxidizing archaea(AOA)to the combined application of biochar and nitrogen fertilizer，soil culture experiment with different amounts of biochar and nitrogen fertilizer was conducted with the soil that had been planted with coffee for more than ten consecutive years. The results showed that the combined application of biochar and nitrogen fertilizer alleviated the decrease in soil pH，reduced the content of exchangeable Al3+ in the soil，and significantly increased the total organic carbon content. The relative abundance of AOB was higher than 57%，which was the main ammonia-oxidizing microorganisms. The total number，diversity，and richness of AOB and AOA were significantly increased by biochar application combined with nitrogen fertilizer，with the best effects observed at a combined application of 10% biochar by dry soil weight and nitrogen fertilizer. Biochar could reduce soil acidity and enrich the diversity and community structure of soil ammonia-oxidizing microorganisms. Thus，the combined application of biochar and nitrogen fertilizer should be given priority in practical production.