DNAzymes have been widely and effectively used for the detection of pathogenic bacteria, which pose a serious public health threat. However, the rapid and cost-effective detection of such bacteria remains a major challenge. In this study, we successfully selected Vibrio alginolyticus-specific DNAzymes. The activity of the candidates was assessed via fluorescence intensity and gel electrophoresis. The DNAzyme DT1 had a detection limit of 31 CFU/ml for V. alginolyticus and exhibited high specificity.Graphene oxide (GO) was used to develop a DNAzyme-based fluorescent sensor for the detection of V. alginolyticus, which significantly improved detection performance and shortened the reaction time as little as 10 s. The proposed method was then validated using crab, shrimp, fish, clam, and oyster samples. This study thus provides a new method for the rapid and sensitive detection of V. alginolyticus.