The study investigated the effects of soybean oligosaccharides (SBOS) on the microbial community structure within a biofloc system and identified the optimal concentration. The control group utilized glucose as carbon source, whereas experimental groups replaced it with 2.5%, 5.0%, 7.5%, and 10.0% SBOS, respectively. Ammonium chloride served as the nitrogen source with C/N ratio of 15, and Bacillus subtilis was introduced to establish the biofloc system. The results indicated that total nitrogen, ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen concentrations in the water were reduced, with no significant differences observed among all groups (P>0.05). High-throughput 16S rRNA sequencing revealed that Proteobacteria was the dominant phylum at 14 days in both experimental and control groups. By 28 days, Proteobacteria and Bacteroidota proportions were similar. At the genus level, Bacillus abundance significantly increased in the SBOS-2.5 and SBOS-10.0 groups at 14 days (P<0.05), while Aeromonadaceae abundance significantly decreased (P<0.05). At day 28, Flavobacterium abundance significantly increased in the SBOS-2.5 and SBOS-10.0 groups compared to the control group (P<0.05). Additionally, Chryseobacterium abundance in all experimental groups was significantly lower than control group (P<0.05). Some bacteria may be crucial in the nitrification and denitrification of nitrogen in water, according to a correlation analysis between bacterial composition and environmental conditions. In conclusion, substituting glucose with 2.5% and 10.0% SBOS as the carbon source in bioflocs effectively enhanced the abundance of beneficial bacteria and reduced that of harmful bacteria, thus promoting the stability of bacterial communities in water. Considering experimental outcomes and application costs, a 2.5% SBOS replacement ratio for glucose is recommended. This study provides a foundation for the practical application of SBOS as a carbon source in the biofloc systems and offers insights for future research.