Aiming at the problem of how to achieve the rapid detection of pathogenic microorganisms, this paper takes tobacco ringspot virus as the detection object, designs the impedance detection and analysis platform of tobacco ringspot virus based on microfluidic impedance method, establishes an equivalent circuit model to analyze the experimental results, and determines the optimal detection frequency of tobacco ringspot virus detection. Based on this frequency, an impedance–concentration regression model was established for the detection of tobacco ringspot virus in a tobacco ringspot virus detection device. Based on this model, a tobacco ringspot virus detection device was designed by using an AD5933 impedance detection chip. A comprehensive test study was carried out on the developed tobacco ringspot virus detection device through various testing methods, which verified the feasibility of the tobacco ringspot virus detection device and provided technical support for the field detection of pathogenic microorganisms.