The invention provides a fabrication method of an electrochemical immunosensor for detecting brucellosis, and belongs to the technical field of an electrochemical biosensor. The method comprises the steps of pre-processing a glassy carbon electrode, modifying a gold nanoparticle onto the glassy carbon electrode by a method of electrodeposition, introducing glutathione as a dirt-resistant material,self-assembling with a gold surface, effectively rejoining HA onto the glutathione by N-(3-dimethy inaminoproyl)-N'-ethyl carbodiimide and N-hydroxysuccinimide, and finally, enclosing a non-speical active site by erythritol. By the method, the problems of poor accuracy, complicated processing process and the like of an existing brucellosis detection method are solved. An electrode interface of the immunosensor has excellent hydrophilcity, the pollution-prevention property of a composite interface is obviously improved, the method has the advantages of ultrahigh sensitivity, relatively wide linear range, relatively low detection limit, good stability and repeatability, is simple and convenient to operate and can be used for detecting a brucellosis antibody in a complicated biological sample.