The invention relates to immune colloidal gold test paper for detecting listeria hemolysin and a preparation method thereof, belonging to the field of biotechnology. In the invention, firstly, the listeria hemolysin expressed by the recombinant Escherichia coli is purified, and then, the purified listeria hemolysin is used for preparing polyclonal antibodies and monoclonal antibodies. Colloidal gold is used for marking the monoclonal antibodies as tracers, anti-hemolysin polyclonal antibodies and anti-mouse polyclonal antibodies are respectively coated in the corresponding positions of a detection line and a quality control line, and the anti-hemolysin polyclonal antibodies and the anti-mouse polyclonal antibodies can be specifically combined with corresponding antigens to be trapped and reddened. The negative and positive results can be judged according to whether the detection line and the quality control line develop or not. The invention has the characteristics of simple and convenient sampling, strong specificity, high sensibility and the like, can conveniently, quickly and accurately detect the listeria hemolysin in 5min, and can also be used for detecting whether samples of animals, animal tissues, silage, foods, meat products, excrement and the like are polluted by the listeria capable of generating the hemolysin.