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κ-casein (κ-CN) plays an important role in the stability and coagulation properties of milk. An immunoassay to quantify κ-casein in milk, using an optical biosensor based on surface plasmon resonance (SPR) measurement, has been developed. The assay consists of a two-step sandwich strategy, with two anti-κ-casein antibodies directed against each extremity of the casein to quantify only native κ-casein. The analysis time per sample was less than ten minutes. The antibody-coated surface could be used for more than 150 determinations. The detection limit was established at 0.45 μg.ml−1 and the intra- and inter-assay variation coefficients were 4.28% and 6.8%, respectively. The method was applied to raw milk in order to quantify intact κ-casein, with no pretreatment of the sample. It also allowed the monitoring of κ-CN concentration during milk coagulation after addition of rennet.