Biofloc technology (BFT) is an advanced aquaculture method that uses microbial communities to enhance water quality and support aquatic species cultivation. Our research aims to delve into the pivotal role of aeration intensity within BFT systems, revealing its influence on microbial community structures, water quality, and nutrient cycling for L. vannamei culture. Three aeration levels were set with intensities of V75 (75 L/min), V35 (35 L/min), and V10 (10 L/min). The results showed that the lowest aeration intensity (V10) resulted in larger floc sizes and a reduction in the 2D-fractal dimensions, indicating a decreased overall structural complexity of the bioflocs. In addition, water quality parameters, including total ammonia nitrogen and nitrite, remained low across all treatments, highlighting the water-purifying capacity of biofloc. While protein and lipid contents in biofloc did not differ significantly among treatments, docosahexaenoic acid (DHA) levels were highest in the V75 treatment, suggesting that higher aeration promotes the accumulation of essential fatty acids. RDA analysis revealed that microorganisms like Ruegeria sp. and Sulfitobacter mediterraneus negatively correlated with ammonia and nitrite levels, suggesting their key role in converting ammonia to nitrite and nitrate in marine nitrogen cycles. The functional annotation of metagenomes across different aeration levels showed the similarly active roles of microorganisms in nitrogen metabolism and protein synthesis. In conclusion, while variations in aeration intensity affect floc size and the accumulation of essential fatty acids in biofloc, they do not significantly impact overall water quality or core microbial functions in L. vannamei aquaculture. Future research should focus on the effects of aeration strategies on microbial community dynamics and the integration of these data with performance metrics in L. vannamei. These insights can help optimize biofloc cultivation and enhance environmental sustainability in the aquaculture industry.