Surface plasmon resonance (SPR) is a powerful label-free diagnostic tool to study biomolecular interactions. However, one of the drawbacks of SPR is the lack of controlled immobilization of ligands on the sensor surface. We have developed a modular platform for the fast, reagent-free and site-specific immobilization of azide-containing ligands by strain-promoted cycloaddition onto a cyclooctyne-modified SPR sensor surface. The usefulness of the concept was shown in a study with a papain model system, and up to 150 experiments were performed without loss of surface quality. Furthermore, azide-containing green fluorescent protein (GFP) was also effectively immobilized. Taken together, cyclooctyne-modified SPR chips enable smooth and site-selective immobilization of ligands and prove to be more robust than traditionally functionalized systems.