[Objective] The aim of this study was to check the effect of water quality control by RAS (Recirculating Aquaculture System) and analyze the function of microbial community on different substrates during the period of Microbrachium rosenbergii over-wintering cultivation. [Methods] The microbial samples were collected from pond water, artificial aquatic plants which were composed of normal fiber membrane and nano fiber membrane in external biofilter after RAS being operated for 88 days. The V4 and V5 regions of 16S rRNA gene on three different substrates microbial were analyzed using DNA extraction, PCR amplification and quantification, high-throughput miseq sequencing technology. Sequence data was processed by read trimming and identification of V4?V5 sequences, followed by filtering and assigning the operation taxonomic units (OTU). Based on the OTU analyzed the microbial community diversity index and structure. Water quality was monitored every 3?4 days with the national standard method. [Results] The pond water quality maintained in a good condition that ammonia-nitrogen and nitrite-nitrogen kept at 0.17±0.08 mg/L and 0.28±0.15 mg/L, respectively. Bacteria composition and community diversity varied in three substrates and 64 species were identified which belonged to 64 genus and 9 phylum including Proteobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Planctomycetes, Nitrospirae, Acidobacteria and Chlorobi. On the level of genus, Comamonadaceae_unclassified was the most dominant genus in pond water which both appeared in the other two substrates. Inhella dominated in surface of normal fiber while Fusibacter was predominant in nano fiber membrane surface. Nano fiber membrane had the highest bacteria community diversity, followed by normal fiber membrane and then pond water. Water quality of over-wintering cultivation maintained at a relatively steady state after operating recirculating aquaculture system for 40 days. [Conclusion] It is viable to regulate and control water quality of over-wintering cultivation by using fiber membrane as a microbial substrate. With the development of new material science, it is necessary to develop a filter material suitable for aquaculture.