The invention relates to a colloidal gold detection card for rapidly detecting rabbit plague virus and a preparation method thereof. The specific steps are: (1) preparation of paired anti-rabbit plague virus monoclonal antibodies, including immune procedures, cell fusion, hybridoma screening and Cloning, antibody preparation and purification; (2) colloidal gold detection card for rapid detection of rabbit plague virus and its preparation method, including preparation of colloidal gold monoclonal antibody, sample pad treatment, spray film, C and T line determination, tested Sample processing, performance measurement. The invention can quickly, sensitively and accurately detect rabbit plague virus infection in the liver and kidney of rabbits, overcomes the domestic method of detecting rabbit plague virus mainly by red blood cell agglutination test, greatly shortens the detection time, and provides convenience for on-site detection.