Abstract: In order to establish an indirect competitive ELISA for the detection of ofloxacin (OFLX) residues in honey, in this study, OFLX was coupled to carrier proteins using the active ester method to obtain anti-OFLX complete antigen (OFLX-BSA) and detection antigen (OFLX-OVA). BALB/c mice were immunized with OFLX-BSA, after which anti-OFLX monoclonal antibodies were prepared by hybridoma and other techniques. An indirect competitive ELISA method was developed by optimizing the reaction conditions and the accuracy, precision and specificity of the method were determined. The results showed that OFLX was successfully coupled to the carrier protein, a hybridoma cell line (3D7) against OFLX was obtained. And the IC₅₀ value of the monoclonal antibody was 1.17 ng/mL. The average recovery rate of OFLX spiked in honey by this method was 84.1%, and its intra-batch coefficients of variation were greater than inter-batch ones in all batches. The cross-reaction rate with marbofloxacin was 52.9%, and no cross-reaction with the other competing reactants. The indirect competitive ELISA method developed in this study is able to fulfill the requirements for determination of OFLX residues in honey.