The invention discloses a preparation method and application of a rabbit anti-grouper serum immunoglobulin HRP-labeled antibody, and belongs to the technical field of antibody preparation. The rabbit anti-grouper serum immunoglobulin HRP-labeled antibody is an enzyme antibody conjugate comprising HRP and a rabbit anti-grouper serum immunoglobulin antibody, the rabbit anti-grouper serum immunoglobulin HRP-labeled antibody can be specifically bound with grouper serum immunoglobulin for color development of a HRP substrate. The HRP-labeled antibody prepared by the method has high purity, high enzyme activity, suitable conjugate labeling rate, great dilution degree for performing ELISA tests, and high sensitivity, and can be used for detecting viral diseases, parasitic diseases and bacterial diseases of grouper.