Ofloxacin (OFL), a third-generation fluoroquinolone, is widely used as an antibacterial drug to prevent diseases in livestock and poultry. In this study, a paper sensor based on gold nanoparticle and a highly specific monoclonal antibody (mAb) against OFL was prepared to detect OFL residues in beef. The limit of detection of the developed immunochromatographic strip for OFL reached 0.16 ng/mL, and the linear range was from 3.125 ng/mL to 100 ng/mL, with the following optimal parameters for the preparation of colloidal gold-labeled mAb probe: pH 6.5, 2 mg/mL OFL–bovine serum albumin antigen, 10 μg/mL antibody, and 12 min immunoreaction time. Cross-reactivity (CR) experiment indicated that the developed strip is highly specific and features low CR with fluoroquinolone analogues. Overall, the developed strip is reliable for the rapid detection of OFL in beef and can be considered an effective screening method for food safety and quality management

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