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The development of rapid nucleic acid detection methods, which are promising the diagnostic standard of the infectious disease, could expand more options for disease traceability and controllability. Nucleic acid hybridization-based biosensing techniques still encounter limitations in meeting the requirements for rapid detection. Therefore, we proposed a potential-assisted ratiometric electrochemical biosensor for rapid and accurate target DNA detection. This dual-signal analysis system actuated an enzyme-free detection process. The application of an external electric field accelerated molecular dynamics, resulting in highly efficient collision chances. This hybridization process was thus improved from hours to minutes compared with passive hybridization approach. The biosensor not only had a high sensitivity with the detection limit of 12 fM, but also features a robust capability in identifying single-base mismatch. Moreover, the biosensor realized sensitive detection of target DNA in complex biological environments. Overall, this sensing strategy exhibits a promising potential for application in point-of-care testing. Schematic representation of the provided potential-assisted ratiometric electrochemical biosensor for target detection. [Display omitted] • The process of hybridization was finished at electrode interface within 90 s under the application of electric field. • The whole strategy was performed without any use of enzymes. • The potential-assisted electrochemical biosensor has more outstanding sensitivity. • Signal mismatched target was successfully identified, adequately indicating a good specificity. • A great potential in the development of POCT. [ABSTRACT FROM AUTHOR]