Abstract: The aim of the study was to understand the microbial structure in a in-pond three-dimensional culture system (with body mass of 150 g at a stocking density of 11 940 ind./hm2)-Pacific white shrimp Litopenaeusvannamei (with body length of 0.6-1.0 cm at a stocking density of 2 539 000 ind./hm2)-water spinach Ipomoeaaquatica Forsk (at a density of 12 kg per pond). The microbial community structure and composition variation were analyzed in water, sediment, rhizosphere and rhizoplane of water spinach, and intestine of river puffer and Pacific white shrimp in the system by means of Illumina MiSeq sequencing to detect two hypervariable regions (V3 and V4) of bacterial 16S rRNA. The six groups of bacteria in the system were distributed in 62 phyla, and 1 503 genera, with dominant phyla including Proteobacteria (27.71%), Actinobacteria (16.37%), Cyanobacteria (16.05%) and Bacteroidetes (10.07%). Rhodococcus, Staphylococcusg, Arcobacter, Rhodobacter, Thiobacillus and Mycoplasma were the dominant genera in the ponds, Actinomycetes, Proteobacteria, Cyanobacteria and Bacteroidetes were the dominant bacterial phyla in the water, Proteobacteria and Bacteroidetes were the dominant bacterial phyla in the sediment. Proteobacteria, Cyanobacteria and Bacteroidetes were the dominant bacterial phyla in rhizosphere and rhizoplane of water spinach, Firmicutes, Epsilonbacteraeota, Cyanobacteria and Bacteroidetes were the dominant bacterial phyla in the intestine of river puffer, and Actinomycetes and Proteobacteria were the dominant bacterial phyla in the intestine of Pacific white shrimp. There was slightly different distribution of functional bacteria in the in-pond three-dimensional culture system. There was significantly higher relative abundance of nitrobacteria in water spinach rhizoplane than that in water and the intestine of river puffer and Pacific white shrimp (P<0.05). There was significantly higher relative abundance of denitrobacteria in water spinach rhizosphere than that in the other parts (P<0.05), without significant difference among other components (P>0.05). The relative abundance of azotobacter was shown to be significantly higher in water and rhizosphere than that in the intestine of Pacific white shrimp and river puffer, and sediment (P<0.05). The microbial community structure was quite different in different components including sediment, water, water spinach rhizosphere and rhizoplane, and intestine of river puffer and Pacific white shrimp in the system due to different functions. High relative abundances of nitrobacteria, denitrobacteria and azotobacter were observed in rhizosphere and rhizoplane, which promoted the nitrogen cycle and improved the nitrogen utilization in the system. Meanwhile, there were both beneficial bacteria and pathogenic bacteria in the intestine of river puffer and Pacific white shrimp.