Abstract: In this study, nitrifying biofloc was cultivated from sewage outlet sediment by gradually reducing carbon source so as to improve its nitrogen removal performance, reduce the cost and shorten the culture period. High throughput sequencing technology was used to analyze the bacterial community structure of the seed sludge and nitrifying biofloc. The results show that the main dominant flora of seed sludge was consistent with other reported heterotrophic flocs, indicating that the sewage outlet sediment has good microbial flora foundation and can form nitrogen removal stable biofloc within 7 d. As the decrease of carbon source, the microbial flora structure of heterotrophic biofloc changed, and the nitrifying biofloc gradually formed after 32-day target training. The dominant bacterial communities of the seed sludge and nitrifying biofloc were Proteobacteria and Bacteroidetes. At Class level, the dominant flora of seed sludge were Gammaproteobacteria, Bacteroidia and Deltaproteobacteria, while the dominant flora of nitrifying biofloc were Bacteroidia, Gammaproteobacteria and Anerolineae. Among them, the total relative abundance of nitrifying bacteria in nitrifying biofloc had greatly increased compared with the seed sludge. The nitrifying biofloc contributes to high nitrogen removal performance and less aquaculture cost.