The effects of system temperature and DO level in aerobic reactor on nitrification and denitrification in three lab-scale moving bed biofilm reactors(MBBRs)in series operated under anoxic/aerobic/aerobic conditions respectively, were investigated. The microscopic mechanism based on the changes in the structure of nitrogen removal functional bacteria in biofilm affected by temperature and DO were discussed using high-throughput sequencing technology. The results indicated that the increase in system temperature could enhance not only nitrification but also denitrification process in biofilm, and the increase in the DO level in aerobic reactor was beneficial to nitrification process. During the continuous operation of the processes, when the system temperature and the DO level in the aerobic reactor were at the highest level (i.e., temperature=20~22℃, DO=5~8mg O₂/L), more than 1.60g NH₄⁺-N/(m²·d) of the specific nitrification loading rate could be achieved, and the specific denitrification loading rate was as high as 2.84g NO₃^--N/(m²·d). The optimal removal efficiencies of NH₄⁺-N and TN could reach 98.7% and 85.7% respectively. The essential reasons for the changes in the population and community structure of nitrogen removal functional bacteria resulted from the variation in temperature and DO level. When the DO level in the aerobic reactor decreased, the OTUs proportion of nitrifiers, especially heterotrophic nitrifiers, decreased significantly. The effect from the temperature on the denitrifiers could be mainly attributed to the structural change in the community.