Eutrophication due to aquaculture can cause the decline of seagrasses and impact their carbon storage capacity. This study explored the effects of aquaculture on the sediment organic carbon (SOC) in Thalassia testudinum seagrass beds using enzyme activity and microorganisms as indicators. Our results showed that the distance to aquaculture significantly increased the SOC and TN of sediments; the C/N ratio of sediments was reduced by the distance to aquaculture. Distance to aquaculture and seagrasses significantly impacted the δ13C of sediments, and their significant interactive effects on the δ13C of sediments were found. Distance to aquaculture and seagrasses had significantly interactive effects on the cellulase activity of sediments. Distance to aquaculture and seagrasses separately reduced the invertase activity of sediments. SOC in the seagrass bed was significantly positively impacted by cellulase activity and polyphenol oxidase activity in sediments. Firmicutes, Desulfobacterota and Chloroflexi were the dominant taxa in the S1 and S2 locations. From the S1 location to the S2 location, the relative abundances of Firmicutes and Desulfobacterota increased. The functional profiles of COG were relatively similar between the S1 and S2 locations. BugBase phenotype predictions indicated that the microbial phenotypes of all the seagrass sediment samples were dominated by anaerobic bacteria in terms of oxygen utilizing phenotypes. FAPROTAX functional predictions indicated that aquaculture affects functions associated with seagrass bed sediment bacteria, particularly those related to carbon and nitrogen cycling. This study can provide an important basis for understanding the response mechanism of global carbon sink changes to human activities such as aquaculture and supply more scientific data for promoting the conservation and management of seagrass beds.