Large-scale cultivation of Gracilaria lemaneiformis developed in the Nan’ao area, Shantou, Guangdong Province in 2005. Nan’ao Island is an important base for Gracilaria cultivation in China. This survey was conducted in a re-planning culture area of the G. lemaneiformis cultivation system in Nan’ao. Samples of water, sediment, cultivated Gracilaria, and their litter were collected from the seaweed cultivation area (G). The current Gracilaria cultivation area is a mixed cultivation area of shellfish and algae, where oyster culture has been performed for many years. Samples were also collected from the adjacent control area (C) when the seaweed exhibited the largest biomass. The results showed that the amounts of bacteria were 3.96×10⁵ copies/mL and 4.97×10⁵ copies/mL, respectively, in the surface and bottom water of the G area. They were significantly higher than that in the C area. However, sediment bacteria showed no significant differences between the G and C areas. The densities of sequenced bacteria and cultured bacteria on the Gracilaria surface were significantly higher than that in their litter. Among different habitats (water, sediment, and G. lemaneiformis), bacterial diversities of the two areas were higher in the sediment. Compared with the C area, the relative abundance of Bacteroidetes and Firmicutes was higher in the sediment of G area. For water samples, the dominant bacteria were similar between those two areas, but a higher relative abundance of Rhodobacteraceae was observed in the G area. The main dominant genus attached to Gracilaria and their litters were Geitlerinema, Vibrio, and Pseudoalteromonas. Correspondingly, Vibrio alginolyticus, Vibrio cyclitrophicus, and Pseudoalteromonas sp. were isolated and identified among the culturable dominant bacteria. Through the redundancy analysis (RDA), pH, DO, and salinity were the dominant factors affecting bacterial community compositions in water and on the surface of G. lemaneiformis. Total phosphorus (TP) was the dominant factor affecting bacterial community composition in sediments. The results indicated that large-scale cultivation of G. lemaneiformis plays an important role in the assembly of bacterial communities in both the water body and sediment in the seaweed cultivation ecosystem. Furthermore, the surface of cultivated Gracilaria and its litter had specific microflora, which may affect the structure and function of the seaweed cultivation ecosystem.