Abstract: The closed shrimp farming model based on bioflocs effectively isolates external environmental microorganisms, such as pathogenic microbes, from interfering with the aquaculture water environment. Simultaneously, it enables targeted manipulation of the microbial community within this system through methods like probiotic supplementation. Therefore, identifying the key microorganisms in this model, determining regulatory methods, and revealing the succession patterns of its microbial community are crucial for achieving targeted microbial community manipulation. This study employs microbial diversity analysis and metagenomic analysis to investigate microbial community succession patterns within the biofloc-based closed shrimp farming system and identify its key functional genes. By analyzing water quality dynamics in relation to environmental factors, the study identified critical factors influencing microbial communities and pinpointed key microorganisms for water quality improvement. Results indicate that microbial community succession in this system is influenced by ammonia nitrogen and nitrite concentrations in the water, with trends in microbial diversity indices largely consistent with nitrite concentrations. Among these, the abundance of bacteria belonging to the genus TM7a within the order Saccharimonadales showed a highly significant positive correlation (p < 0.001) with nitrite concentration, suggesting they may be key microorganisms involved in denitrification. Additionally, the OTU3181 strain within this order exhibited a highly significant positive correlation with ammonia nitrogen concentration during the later aquaculture phase (p < 0.001), suggesting it may be a key microorganism for ammonia nitrogen removal during this period. Metagenomic metabolic analysis revealed a high proportion of denitrification and dissimilatory nitrate reduction (DNRA)-related genes in the biofloc, suggesting these represent the primary nitrogen metabolic pathways in this system. The high correlation between aquatic environmental conditions and dominant microbial communities indicates that the evolutionary trajectory of microbial communities can be artificially guided by regulating key environmental factors and introducing specific bacterial strains. This research offers direct applications for cultivating efficient biological flocs for water purification in shrimp farming and facilitates the targeted microbial reconstruction of these systems.