AbstractVibrio cholerae(Vc) causes cholera disease. Vc contamination is widely found in water and aquatic products, and therefore is a serious food safety concern, especially for the seafood industry. In this paper, we attempted the rapid detection ofV. cholerae. Nine rounds of in vitro selection using an unmodified DNA library were successfully performed to find specific DNAzymes of Vc. Their activity was evaluated based on a fluorescence assay and gel electrophoresis. Finally, a DNAzyme (named DVc1) with good activity and specificity with a detection limit of 7.2 × 103CFU/mL of Vc was selected. A simple biosensor was constructed by immobilizing DVc1 and its substrate in shallow circular wells of a 96‐well plate using pullulan polysaccharide and trehalose. When the crude extracellular mixture of Vc was added to the detection wells, the fluorescent signal was observed within 20 min. The sensor effectively detected Vc in aquatic products indicating its simplicity and efficiency. This sensitive DNAzyme sensor can be a rapid onsite Vc detection tool.