Abstract: Objective To establish a real-time PCR Taqm an assay for the detection of Vibrio fluvialis.Methods The conserved region of tox Rgene of V.fluvialis was used to design primers and Taqman probe.The real-time PCR Taqm an system for V.fluvialis detection was established and the concentration of primers and probe were optimized.The sensitivity and specificity of the assay were evaluated and compared with PCR.Results The real-time PCR Taqman system for V.fluvialis detection was established.The concentration of primers and probe in the assay was 100 nmol/L and 200 nmol/L, respectively.The sensitivity of the real-time PCR Taqman system to detect pure bacterial was 4.17×10² cfu/mL and was 1000 times higher than that of PCR.No amplification of other bacterium was observed in the templates when targeting tox R gene of V.fluvialis was detected.Conclusion The established assay can detect V.fluvialis sensitively and specifically and is a sensitive and rapid way to detect V.fluvialis.