The highly conserved region of toxR gene of Vibrio rotiferianus was selected as the target fragment for the specific primer design. The recombinant plasmid was constructed based on the primers and we established a fluorescence quantitative PCR method for V. rotiferianus. And UF-150 Genechecker microfluidic quantitative real-time PCR instrument was used as a platform to develop a microfluidic quantitative real-time PCR detection method for V. rotiferianus via the optimized reaction system and reaction conditions. The results indicated that, this method can specifically amplify toxR gene target fragment, and the lower detection limit for V. rotiferianus is 1.34×10⁰ copies/μL. Artificial infection detection results show that the lower detection limit for V. rotiferianus in fish tissues is 1.34×10³ CFU/g. The results can be visually interpreted and the detection time was less than 42 min. This method has prominent advantages of strong specificity, high sensitivity and low site requirements, and it is suitable for developing practical field rapid detection technology of aquatic pathogens.