Aiming at the problem that the detection of aquaculture pathogens suffers from difficult isolation, time-consuming and low degree of automation, a detecting method of aquaculture pathogens concentration based on microfluidic system with magnetic isolation was proposed．A dedicated microfluidic chip with automatic sample injecting, magnetic isolation and impedance measurement was designed, and the experiment platform based on microfluidic detection system was built. The optimum impedance measurement frequency, magnetic force and target bacteria capture time in the microfluidic system were determined, and as an example of Escherichia coli O157:H7 the performance of the system was experimentally verified. Experiment results showed that compared with plate counting method the proposed method was nearly the same in detection accuracy, and the detection time was decreased from 48 h to 50 min because of no-cultivation. The microfluidic system realized rapid isolation and detection for aquaculture pathogens with increased automation in the prevention of aquaculture disease.