A highly sensitive and specific monoclonal antibody (Mab) against diclazuril was produced. The hapten with diclazuril coupled to diazotised 4-aminobenzoic acid was synthesized and conjugated to bovine serum albumin by the active ester method to form an immunogen for antibody generation. A novel diclazuril carboxymethyloxime derivative used in an ovalbumin conjugate was applied as a heterologous coating antigen and was expected to improve the immunoassay sensitivity. A sensitive and simple indirect competitive enzyme-linked immunosorbent assay (icELISA) based on the Mab for the determination of diclazuril was developed. Under the optimized conditions, the icELISA for diclazuril showed a half maximum inhibition concentration (IC50) value of 1.8 ng/mL, with limit of detection of 0.24 ng/mL and negligible cross-reactivities with other coccidiostat compounds including toltrazuril, robenidine, nicarbazin, halofuginone, amprolium, monensin, and maduramycin. The icELISA was successfully applied to diclazuril residue analysis in spiked chicken tissues. The average recoveries, intra-assay, and inter-assay coefficients of variation were in a range from 77.6 to 103.7 %, 3.7 to 13.0 %, and 5.6 to 18.3 %, respectively.