Due to its ability to collect and remove aquaculture waste, the in-pond raceway system (IPRS) has changed the uncontrolled waste discharge in the traditional cage aquaculture method in applying large water bodies. However, when applied to large water bodies, its environmental performance is still lacking. This study focused on the comparative analysis of the microbial characteristics and the interaction between each interface of the IPRS (fish gill and gut, water and sediment). Further, it revealed the primary relationship from the perspective of microbiota in the IPRS.