A competitive indirect chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for florfenicol (FF) and its major metabolite florfenicol amine (FFA) residues in chicken muscle has been developed and validated according to Commission Decision 2002/657/EC criteria. The IC₅₀ value of the method was 0.153 μg kg⁻¹ for FFA with a cross-reactivity of 74.3% for FF under optimum conditions, in which FFA–F–BSA (FFA–formaldehyde–BSA) and FF–G–OVA (FF–glutaric anhydride–OVA) were used as an immunogen and a coating antigen, respectively. FFA and FF were easily extracted from chicken muscle with a 40 : 1 ethyl acetate–ammonia mixture, obtaining recoveries of 70.3–100% (FFA) and 71.8–102.0% (FF). Accuracy, precision, selectivity, robustness, limit of detection (LOD), limit of quantification (LOQ) and detection capability (CCβ) of the assay have been assessed during the validation process. LOD values in chicken muscle were 0.353 μg kg⁻¹ for FFA and 0.526 μg kg⁻¹ for FF (10-fold dilution) and 0.453 μg kg⁻¹ for FFA and 0.657 μg kg⁻¹ for FF (100-fold dilution). Furthermore, the CL-ELISA method gave CCβ values of 1.0 μg kg⁻¹ for FFA and FF. Finally, real chicken muscle samples were analyzed with the CL-ELISA method, traditional ELISA and a previously reported gas chromatography-negative chemical ionization mass spectrometry (GC-MS), and results confirmed the utility of this new CL-ELISA for trace determination of FF and FFA, simultaneously.