We report on a novel covalent coupling method using electrochemical activation of hydroquinone monoester self-assembled monolayers. The reaction generates benzoquinone as a good leaving group, followed by nucleophilic acyl substitution with a primary amine to form an amide in high yield. The method allows the site-selective and the reaction-controlled positioning of biotin on the individually addressable microelectrode array and, subsequently, density-differentiated patterning of streptavidin on the biotin surfaces. Because the electrochemical coupling method provides a very rapid, mild, and quantitatively controllable reaction pathway for covalent bond formation on organic surfaces, it will be used as a versatile molecular anchoring tool in fields such as molecular electronics and biochip technology.