An enzyme-linked immunosorbent assay (ELISA) for analysis of fluoroquinolones residues in animal feeds has been developed and validated according to Commission Decision 2002/657/EC criteria. Initially, direct and indirect competitive ELISA formats were compared for one fluoroquinolone polyclonal antibody (As172) and two monoclonal antibodies (FQ8 and FQ10), in order to find the best combination in terms of simplicity, reduction of matrix effect and sensitivity. The optimal methodology was identified as direct ELISA format using polyclonal antibody As172, able to avoid the matrix effect by only 10-fold dilution of feed samples. Following the optimized ELISA protocol, the half-maximal inhibitory concentration (IC50) and limit of detection (LOD) for enrofloxacin was determined to be 15.2 ng·g−1 and 1.3 ng·g−1, respectively. Decision limit (CCα) obtained was 10 ng·g−1 and detection capability (CCβ) was 20 ng·g−1. Significant cross-reactivity values (>42%) were obtained for eight fluoroquinolones by the optimized ELISA method. Moreover, comparison of results from ELISA to that of liquid chromatography with fluorescence detection (LC-Fl) showed good correlation. In general, the developed ELISA allows a rapid, sensitive, and low-cost screening analysis of fluoroquinolone residues in animal feeds.