The invention relates to an enzyme immunosensor based on chitosan and nano-gold, and by using the specific combination of the monoclonal antibody of the cell wall of mycobacterium trberculosis with mycobacterium trberculosis, the enzyme immunosensor is applicable to the rapid detection of mycobacterium trberculosis in milk. The characteristic is that the enzyme immunosensor for detecting mycobacterium tuberculosis is prepared by the following steps: modifying the surface of a glassy carbon electrode through electrochemical deposition method by a mixed solution of chitosan gel, nano-gold solution, and goat anti-mouse antibody labeled by alkaline phosphatase; and fixing the mycobacterium trberculosis monoclonal mouse antibody on the surface of the glassy carbon electrode modified by the goat anti-mouse-chitosan-nano-gold film which is labeled by alkaline phosphatase through the specific antigen-antibody reaction. Quantitative analysis is performed by detecting the electric signal changegenerated before and after the incubation reaction of mycobacterium trberculosis and the modified electrode through differential pulse voltammetry. The preparation method of the sensor is simple, andthe sensor has the advantages of high sensitivity, short detection time, and simple operation, and is applicable to the method for the rapid detection of mycobacterium trberculosis.