An assay for quantification of riboflavin (Rf) in milk-based products has been developed using the principle of surface plasmon resonance with on-chip measurement. The quantification was done indirectly by measuring excess of Rf binding protein (RBP) that remains free after complexation with Rf molecules originally present in the sample solution. The chip was modified with covalently immobilized Rf in order to bind the RBP in excess. A chemical modification was performed to introduce a reactive ester group at the N-3 position of the natural Rf to bind amino groups present on the chip surface. Calibration solutions were prepared by mixing a range of Rf standard solutions with an optimized concentration of RBP. The Rf content in the milk-based products was then measured by comparison of the response against the calibration. Results obtained were very close to those from an official HPLC-fluorescence procedure. The limit of quantification was determined to 234 microg/L and the limit of detection to 70 microg/L by an injection volume of 160 microL.