To establish a rapid assay for Vibrio vulnificus (V. vulnificus) detection, a quantitative real-time PCR (qRT-PCR) method was developed targeting vvhA gene of V. vulnificus . The results showed that the tests for 15 strains of different bacteria by using this method, only the ones of V. vulnificus were positive, indicating high specificity of qRT-PCR. Moreover, the sensitivity of this specific method was 15 CFU/mL. Further stability and reproducibility test results showed that the coefficient of variation of Ct values of four duplicates were less than 2%. In addition, a total 2 positive samples for V. vulnificus were detected from 156 clinical samples by this method, and the results were in accordance with those by SN/T 1870-2007 standard detections. Therefore, the qRT-PCR method provides a novel rapid and sensitive detection method for V. vulnificus infection.