Florfenicol (FF) is a synthetic antibiotic with a broad antibacterial spectrum and the high therapeutic effectiveness that has been developed specifically for veterinary use. Obviously, FF adulterated in animal supplies is one of essential global concerns. A competitive ELISA for the detection of florfenicol in food of animal origin (swine, chicken, and fish) is described. Influence of immunoconjugate structure on the assay sensitivity and specificity was investigated. The new ELISA showed much lower than the MRPLs for FF at 100-3,000 mg kg(-1) in the European Communities and the sensitivity of our ELISA method was superior to that described in other reports. According to the test preparation record, the limit of detection of the developed ELISA performed on meat species was 0.3 µg kg(-1) (IC50 value 1.9 µg kg(-1)). The method developed permits FF concentrations to be determined in the range 0.3-24.3 µg kg(-1). A low cross-reactivity with florfenicol amine (FFA), thiamphenicol (TAP), and chloramphenicol (CAP) was displayed (16.2%, 9.5%, and 9.4%, respectively). Recovery in different food samples (swine, chicken, and fish) averages between 87-115%. The method can be applied for inspection of animal supplies for trace florfenicol residues.