This paper presents a new label-free optical method to study biomolecular interactions in real time at the surface of an optically transparent substrate. The method relies on the change in the absorbance spectrum of a self-assembled monolayer of colloidal gold on glass, as a function of biomolecular binding to the surface of the immobilized colloids. Using this approach, we demonstrate proof of principle of a label-free optical biosensor to quantify biomolecular interactions in real time on a surface in a commercially available UV-visible spectrophotometer and of a colorimetric end-point assay using an optical scanner. The spectrophotometric sensor shows concentration-dependent binding and a detection limit of 16 nM for streptavidin. The sensor is easy to fabricate, is reproducible in its performance, has minimal technological requirements, namely, the availability of an UV-visible spectrophotometer or an optical scanner, and will enable high-throughput screening of biomolecular interactions in real time in an array-based format.