An inexpensive and portable surface plasmon resonance (SPR) sensor, SPReeta Evaluation Kit SPR3, has been used to develop a biosensor for the determination of fluoroquinolone antibiotics (FQs) and to demonstrate its performance analyzing FQ residues in milk samples. The SPReeta three-channel gold chips were activated with a mixed self-assembled monolayer (m-SAM) and functionalized with a FQ haptenized protein. Binding of the antibody produced a concentration-dependent increase of the SPR signal as a result of the change in the refraction index. Similarly, the presence of the FQ produced a dose-dependent decrease of the response, which allowed a good limit of detection (LOD) to be obtained (1.0 ± 0.4 μg L(-1) for enrofloxacin in buffer). The response was reproducible in all three channels, on different injections and days, and also between chips. Milk samples could be analyzed after a simple sample treatment involving fat removal by centrifugation and dilution with water. Under these conditions calibration curves were obtained showing that FQ residues can be analyzed in milk samples with an IC(50) value of 26.4 ± 7.2 μg L(-1) and a LOD of 2.0 ± 0.2 μg L(-1) (for enrofloxacin), far below the European Union regulations for this antibiotic family in this matrix. Finally, the paper also demonstrates that the biosensor is able to selectively detect the presence of FQs in milk samples, even in the presence of other antibiotics. Enrofloxacin, ciprofloxacin, and norfloxacin residues were detected in blind samples supplied by Nestlé Co.