The invention discloses an electrochemical immunosensing method for detecting Listeria monocytogenes, belonging to the field of the biotechnological detection. The surface of a gold electrode is activated electrochemically and then is immersed in 1% of 3-mercaptopropionic acid ethanol solution to be self-assembled to form a monomolecular film, a capture antibody is combined to the surface of the electrode after activation, an enzyme amplification signal is introduced by a sandwich method, and an enzymatic electroactive substance is subjected to oxidation reduction to obtain a current response signal, therefore, a method for detecting the Listeria monocytogenes by use of the electrochemical immunosensor adopting three electrodes as a system is established; the method has good sensitivity, specificity and stability; the detection system is small, the reagent is saved, the detection cost is reduced remarkably, the method has great potential in application and practical sample detection due to no influence from the turbidity of the sample.