This work describes a method for immobilization of biomolecules on surfaces via electrooxidation of surface thiols by generating thiolsulfinates/thiolsulfonates that display a high reactivity toward free thiols in molecules. This is done by applying a potential difference between a platinum cathode and a thiol-derivatized silicon surface as the anode, through a buffer solution. X-ray photoelectron spectroscopy data showed that the amount of thiolsulfinates/thiolsulfonates was voltage dependent. When a potential difference of 1.0 V was applied between the silicon surface and the platinum electrode for 1 min, about 80% of the thiols were converted to thiolsulfonates/thiolsulfinates. The results of comparative chronoamperometric experiments, using a three-electrode potentiostatic setup, indicate that the thiols are oxidized electrochemically at +0.6 V versus Ag/AgCl. This indicates the presence of a direct electrochemical oxidation of the thiols on the silicon surface rather than an oxidation of the thiols by, for example, oxygen formed during the oxidation of water. Peptides containing thiol groups were subsequently covalently bound to the surface through disulfide bonds involving 25% of the thiolsulfinates/thiolsulfonates, which indicates a fully saturated surface coverage with peptide molecules. It was possible to release 40% of the peptides using dithiothreitol, When a voltage was applied to such a surface for a second time, additional thiolsulfinates/thiolsulfonates were formed. This method, used together with nanosized electrodes or a scanning probe microscope tip as a counter electrode, will allow a spatially controlled immobilization of biomolecules on surfaces at the nanometer scale.