The application of biochar is one of the promising management practices to alleviate soil acidification and improve soil fertility. However, it has been found to reduce the content of ammonium nitrogen (NH4+−N) in the soil, which is the most important form of nitrogen (N) for tea tree growth. To investigate the response of soil NH4+−N content to the combined application of biochar and pruned tea plant litter, a pot trial was performed with three treatments: control (CK); biochar (BC); biochar + tea plant litter (BC + L). Soil chemistry properties and ammonification rates were determined, and the microbial community composition was analyzed by high-throughput sequencing. The results showed that the NH4+−N content in BC + L treatment was 1.7–9.5 fold higher than CK and BC treatments after 15 days of application, with no difference in the proportion of ammonia oxidation phyla such as Nitrospirae. The proportion of soil fungus Ascomycota was strongly correlated with the content of soil available nitrogen (p = 0.032), and the relationship was well described by a linear equation (R2 = 0.876, p = 0.01). Further redundancy analysis revealed that soil pH, soil organic carbon (SOC), the ratio of SOC to total nitrogen and the ratio of SOC to alkaline hydrolyzable nitrogen appeared to be important factors influencing the separation of BC + L from CK and BC groups. In summary, the addition of biochar and pruned tea plant litter alters soil properties and may influence the composition of microorganisms with various trophic groups, thus affecting ecosystem function. Our results also highlight the importance of returning pruned materials with biochar application in tea plantation ecosystems.