We report a new approach to surface plasmon resonance (SPR) biosensing based on spectroscopy of multiple surface plasmons on a special multi-diffractive grating coupler. In this approach, the binding event at a surface of the SPR grating coupler is simultaneously observed by surface plasmons of different wavelengths, which makes it possible to determine binding-induced changes in the refractive index distribution at the sensor surface. We demonstrate the potential of this new concept in a model experiment in which three-surface plasmon spectroscopy is used to determine background refractive index variations and changes in the thickness and refractive index of a bovine serum albumin multi-layer grown on the surface of the SPR grating coupler. (c) 2005 Elsevier B.V. All rights reserved.