This paper describes a rapid one-step fluorescence polarization immunoassay (FPIA) for the simultaneous determination of multiple (fluoro)quinolone antibiotics (FQs) in food samples. Several fluorescent tracers were synthesized and evaluated in the FPIA method based on a broad-specificity of monoclonal antibodies toward FQs. The heterogeneous tracer, SAR-5-FAM, was considered as the optimal choice to prepare the immunocomplex single reagent, which allows a rapid and sensitive displacement reaction by addition of analytes. Optimized single-reagent FPIA exhibited broad cross-reactivities in the range of 7.8-172.2% with 16 FQs tested and was capable of determining most FQs at the level of maximum residue limits. Recoveries for spiked milk and chicken muscle samples were from 77.8 to 116%, with relative standard deviation lower than 17.4%. Therefore, this method could be applicable in routine screening analysis of multiple FQ residues in food samples.