Abstract The combination of various evanescent optical methods such as surface plasmon spectroscopy, waveguide mode spectroscopy and an integrated optical Mach–Zehnder-interferometer are used to characterize biotinylated self-assembled monolayers as well as the binding of streptavidin to these labels. The aim of designing a highly specific and sensitive, re-usable affinity sensor for antigens on the basis of an integrated optical Mach–Zehnder interferometer is based on a proper understanding of the characteristics of the entire binding matrix architecture. Therefore, a variety of biotin-derivatives immobilized in a monolayer are investigated with respect to their affinity to streptavidin and the possibility to remove the steptavidin layer specifically. The density of the streptavidin layer as well as the optical constants of the involved molecules are measured. Finally the integrated optical Mach–Zehnder interferometer is tested with respect to the sensitivity to an antigen–antibody binding reaction. An attempt to further increase the sensitivity by simultaneous detection of a fluorescence signal failed due to bleaching effects.