The microbe attached in biofilm carrier material could ulitize different carbon sources in high yield collection pond. In order to understand the metabolic characteristics of these microbes, the Biolog-ECO technology and water quality analysis were used to analyze the change of microbial carbon metabolism and water quality during the formation of biofilm (0-9 w) in high collection pond including grass carp and tilapia. The results showed that:(1) During the test, using biofilm in polyculture ponds has good removal effect of total nitrogen and phosphorus, and has good effects on maintaining water quality stability;(2) For different sampling time points, the metabolic activity of ulitizing carbon source displayed obvious difference:3, 5, 7, 8 w biofilm metabolic activity at high levels; In the biofilm formation process, the trend of the biofilm microbial metabolic activity first rose and then reduced; Richness index, Shannon index, Simpson index, and McIntosh index performance for 3, 4, 5, 7, 8 w were significantly higher than other weeks; (3)Studies on the use of six kinds of carbon sources indicate that biofilm microbes have the highest utilization rates for carbohydrates and polymers; Along with the formation of the biofilm, adherent microbes of glycogen increased obviously, such as n-acetyl-D-glucosamine, beta-methyl-D-glucoside, 1-phosphate glucose utilization of carbon sources; (4) Biofilm microbial metabolic characteristics of principal component analysis (PCA) shows that the different sampling time points of samples gathered in three different areas, including 3, 4, 6, 8, 9 w microbial metabolic difference is small, 0w and 1w as a whole, 5 w and 7 w as a whole, but there were significant differences between the three concentrated areas. The results would provide some reference for further utilization of carbon sources to strengthen biofilm formation in aquaculture.