Abstract: The aim of this study was to investigate the effect of fructooligosaccharides (FOS) on water quality and microbial community in biofloc systems. The control group (CG) was supplied with a mixed carbon source of 70% glucose and 30% starch, while the experimental groups replaced this mixed carbon source with FOS at concentrations of 2.5% (EC2.5), 5% (EC5), 7.5% (EC7.5), and 10% (EC10). The experiments were conducted in the 20 glass aquariums in indoor temperature-controlled system for 28d. Biofloc formation, water quality parameters, and microbial community composition were measured, followed by a joint analysis of water quality and microbial structure. The results showed that: (1) Turbidity and total suspended solids (TSS) in all groups initially increased and then decreased. The turbidity and total suspended solids (TSS) in the EC2.5 group significantly increased on day 14 (P<0.05), whereas the turbidity in the EC10 group significantly decreased (P<0.05). Floc volume (FV) showed pattern of initial increase, followed by decrease, and then an increase, with a peak on day 14. The EC2.5 group had significantly higher FV than that of the control and other experimental groups (P<0.05). (2) Ammonia nitrogen, nitrate nitrogen, and total nitrogen concentrations showed decreasing trend, while nitrite nitrogen initially increased and then decreased, reaching its peak on day 7. On day 7, nitrate nitrogen in the EC2.5 group and nitrite nitrogen in the EC10 group significantly decreased than that of the control group (P<0.05). (3) At the phylum level on day 14, Proteobacteria, Bacteroidetes, and Actinobacteria were the dominant phyla in the CG, EC2.5, and EC10 groups. The abundance of Proteobacteria in the EC2.5 group was significantly higher than that in the control and EC10 groups (P<0.05). At the genus level, the abundance of Flavobacterium and Elstera in the EC2.5 and EC10 groups significantly increased than the control group (P<0.05). (4) On day 14, Verrucomicrobia, unclassified_k__norank_d__Bacteria, and Spirochaetota were significantly positively correlated with nitrate nitrogen at the phylum level. At the genus level, norank_f__37-13 was significantly positively correlated with ammonia nitrogen, while unclassified_c__Alphaproteobacteria showed significantly negatively correlated with ammonia nitrogen. In conclusion, replacing glucose and starch with FOS as carbon source in the biofloc system effectively improves water quality, enhances beneficial bacteria abundance, and optimizes microbial community structure. The recommended FOS concentration of 2.5% is identified. This study provides a theoretical basis for the application of FOS and other functional oligosaccharides as carbon sources in biofloc systems and offers insights for their promotion in aquaculture.