Abstract: To establish a rapid assay for Vibrio alginolyticus(VA) detection,a dual real-time PCR method was developed targeting Collagenase gene of VA. The results showed that the test for 15 bacteria strains of the dual real- time PCR method, only VA test was positive, indicating that the method had high specificity. In addition,the sensitivity of a dual real-time PCR was 18 cfu/m L. Stability and reproducibility of the test results showed that the coefficient of variation of the same sample repeat the test four times Ct values were less than2%. Furthermore,a total 2 positive samples for VA were detected from 165 clinical samples by the real-time method,which was in accordance with the testing result by SN/T 2564-2010 standard detection protocol.Therefore,the real-time method provides a novel rapid and sensitive detection method for VA infection.