A label-free optical biosensor for detection and quantification of diclofenac in bovine milk has been developed. This was achieved by using reflectometric interference spectroscopy as detection method. In a first step, the immunosensor was developed and optimised in buffer concerning sensitivity, selectivity, stability and reproducibility. By comparing recovery rates—not only the good intra- but also the good inter-chip—reproducibility could be proven. Consequently, the assay was transferred in the more complex matrix milk. By utilising an optimised surface modification and evaluation method, matrix effects could successfully be prevented or circumvented. As a result, the developed immunosensor does not need sample pretreatment at all. By obtaining a limit of detection of 0.112 μg L(−1) (0.108 μg kg(−1)), the capability of the developed biosensor is comparable or better than those of standard detection methods. Moreover, the presented biosensor reaches the range of the maximum residue limit (0.1 μg kg(−1)) set by the European Union. Thus, for the first time, diclofenac was successfully quantified at relevant levels in milk by using an optical biosensor.