Objective To establish a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for detecting enrofloxacin (ENR) residues in animal-derived food.Methods ENR was conjugated with keyhole limpet hemocyanin (KLH) using the carbodiimide method to prepare an immunogen, and New Zealand white rabbits were immunized to produce high-sensitivity antibodies. Based on this, the detection system was optimized to establish ic-ELISA, the detection performance was evaluated, and spiked recovery tests were conducted in animal-derived food.Results Under the optimized conditions of coating antigen concentration at 0.25 μg/mL, antibody dilution of 1∶40 000, secondary antibody dilution of 1∶5 000, primary antibody incubation for 15 min, secondary antibody incubation for 30 min, and color development for 5 min, the IC₅₀ of the method was 27.76 pg/mL. The detection range was 0.49~1 577.29 pg/mL, and the limit of detection was 0.33 pg/mL. The intra- and inter-assay coefficients of variation were both less than 15%. The method showed good specificity in trace detection of ENR, with recovery rates of 93.37%~97.06% in chicken and 87.15%~97.59% in fish.Conclusion This method demonstrates excellent sensitivity, precision, specificity, and accuracy, and provides an effective tool for detecting trace residues of ENR in animal-derived food.