Screening of a group of antibiotics from foodstuffs has traditionally relied on sophisticated chemical or physical analysis methods, such as liquid chromatography and mass spectrometric applications. The equipment for these techniques is expensive and not always applicable for high throughput screening. There is a need for an easy and cost efficient detection method for simultaneous screening of structurally similar compounds. Here we describe the engineering of a recombinant antibody which was subjected to oligonucleotide targeted random mutagenesis to emphasize the generic specificity of fluoroquinolone binding. Phage display together with small sized fluoroquinolone derivatives was used to find antibodies of high affinity and generic specificity. The most improved antibody was used to develop a time-resolved fluorescence immunoassay which was further optimized and applied for the detection of fluoroquinolone residues from spiked whole milk samples. The assay can be used to efficiently screen all European Agency for the Evaluation of Medicinal Products (EMEA) controlled fluoroquinolones from whole milk samples with detection levels ranging from 0.2 to 68 μg L(-1).