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In this study, the structural characteristics of the attached microbial community during biofilm formation and the metabolic requirements under anaerobic conditions were evaluated. In particular, 16S rDNA sequencing technology was used to study the structural changes of the attached microbial community during biofilm formation (day 0, 10, 20, and 30) in an aquaculture system, and Biolog microplate technology was used to study metabolic characteristics under anaerobic conditions during biofilm formation. The AWCD (average well colour development), an indicator of carbon metabolism, of attached microorganisms during biofilm formation under anaerobic conditions differed significantly among time points (P < 0.05), and the carbon source utilization capacity was enhanced. Under anaerobic conditions during biofilm formation, the utilization of amines in six major carbon sources was the highest, followed by polymers, carbohydrates, amino acids, and carboxylic acids, and the utilization of phenols was the lowest. Under anaerobic conditions, the utilization rate by the attached microorganisms during biofilm formation was highest for Tween 40, followed by Tween 80, D-xylose, 4-hydroxybenzoic acid, α-D-glucose-1-phosphate, L-phenylalanine, and phenylethylamine. The 16s rDNA sequencing results showed that the dominant phyla of attached microorganisms during biofilm formation were mainly Proteobacteria, Bacteroidetes, Firmicutes, and Cyanobacteria. On the 10th and 20th days of biofilm formation, attached microorganisms were enriched for Rhodobacter of α-Proteobacteria and Janthinobacterium of β-Proteobacteria, which play important roles in biofilm denitrification. However, on the 30th day, enriched bacteria included the Burkholderiales of β-Proteobacteria, the Xanthomonadaceae and Thermomonas of γ- Proteobacteria, which function in cyanide decomposition.