Vibrio species pose a serious threat to human health worldwide. Vibrio parahaemolyticus, V. cholerae, and V. vulnificus capable of causing gastrointestinal infections and sepsis associated with consumption of raw or undercooked seafood are of particular concern. [Objective] To develop a fluorescence quantitative PCR method with improved efficiency and accuracy for the detection of V. parahemolyticus, V. cholerae, V. mimicus, and V. vulnificus. [Methods] The specific primers and probes were designed based on toxR of V. parahaemolyticus and V. mimicus, ompW of V. cholerae, and vvhA of V. vulnificus. A quadruplex fluorescence quantitative PCR method was established by optimizing the reaction system and conditions. [Results] The detection limit of the fluorescence quantitative PCR method was 10 copies/μL, and the amplification efficiency was about 100%. In the specificity test, multiplex real-time fluorescence quantitative PCR and conventional PCR were used to amplify the target bacteria genome, non-target bacteria genome and negative control strain genome, respectively. The results showed that only the target vibrio genome was amplified significantly, indicating that the method had good specificity. In the anti-interference experiment, high concentration Vibrio did not interfere with the detection of low concentration Vibrio. Three repeated experiments were performed for each concentration gradient, and the coefficient of variation of each group was less than 1.5%, indicating that the method was reproducible in this experiment. [Conclusion] The multiplex real-time fluorescence quantitative PCR method was established, which could detect V. parahemolyticus, V. cholerae, V. mimicus, and V. vulnificus specifically and rapidly.