A surface-localized enzymatic AND gate based on scanning electrochemical microscopy was designed and studied. The gate is composed of an insulating glass surface modified with the enzyme glucose oxidase (GOx) and another surface opposing it made of a microelectrode. The latter was modified with a second enzyme, invertase (INV). The distance separating the modified microelectrode and surface controlled the output of the AND gate produced upon the biocatalytic reaction of the confined enzymes. Specifically, as the GOx-modified glass substrate entered the diffusion layer of the microelectrode, it catalyzed the regeneration of an electron-transfer mediator, ferroceniummethanol, generated electrochemically at the tip by oxidizing glucose, also generated at the tip, by catalytic cleaving of sucrose by INV. To enhance the activity of the GOx, mutarotase was added to convert α- to β-glucose to be further consumed by GOx. Hence, an increase of the current at the microelectrode was obtained by approaching the glass surface only in the presence of all the components. This is the first micrometer-sized biomolecular logic gate, of which we are aware, that is surface-confined and shows the promise held by the localization of biomolecular information-processing species.