A hybridoma cell strain secreting a nifursol residue marker monoclonal antibody prepared in the following way: BALB/c mice are subjected to the first immunization with a complete Freund's adjuvant, subjected to booster immunization with an incomplete Freund's adjuvant for four times, and subjected to rush immunization once with nifursol residue marker complete antigen without a Freund's adjuvant so that the BALB/c mice are immunized; the spleen cells of the immunized mice with high titer and low IC50 were fused with mouse myeloma cells by a PEG method, and the fused cells are screened through indirect competitive ELISA and subcloned three times. The monoclonal antibody secreted by this cell strain has good specificity and detection sensitivity (IC50 value of 2 μg/L) to the nifursol residue marker and can be used for residue detection of the nifursol residual marker in food.