[Objective] To establish a simple and rapid molecular typing method for Vibrio parahaemolyticus carrying and non-carrying virulence genes based on molecular motor. [Methods] Four probes specific to virulence genes tdh and trh, species-specific genes tlh and toxR of V. parahaemolyticus were synthesized, and four molecular motor biosensors were constructed by connecting probes to F0F1-ATPase molecular motors through biotin-streptavidin system, respectively. Ten strains of V. parahaemolyticus were classified by the biosensors, and the results were compared with PCR-Electrophoresis-Gel imaging results. Further more, the detection sensitivities and specificities of the molecular motor biosensors were studied. [Results] There were ten strains carrying tdh and none carrying trh, while all ten strains carry tlh and toxR, which was consisitent with the results of PCR-Electrophoresis-Gel imaging. The detection limits of molecular motor biosensors for tlh, toxR, tdh and trh were estimated to be 1 pg/reaction system, and the detection limits of PCR-Electrophoresis-Gel imaging for tlh, toxR, tdh and trh were estimated to be 10 pg/reaction system. The molecular motor biosensors could recognize tlh, toxR, tdh and trh of V. parahaemolyticus specifically. [Conclusion] A molecular typing method was constructed based on molecular motor biosensors and was used to diagnose the pathogenicity of V. parahaemolyticus rapidly and specifically. The detection limits was 10 times higher than those of PCR-Electrophoresis-Gel imaging. The method is easy, rapid, time-saving and labor-saving, especially suitable for the basic laboratories of CDC and port quarantine departments to perform suiveillance and epidemiological traceability of cholera.