A novel glucose biosensor based on capacitive detection has been developed using molecularly imprinted polymers. The sensitive layer was prepared by electropolymerization of o-phenylenediamine on a gold electrode in the presence of the template (glucose). Cyclic voltammetry and capacitive measurements monitored the process of electropolymerization. Surface uncovered areas were plugged with 1-dodecanethiol to make the layer dense, and the insulating properties of the layer were studied in the presence of redox couples. The template molecules and the nonbound thiol were removed from the modified electrode surface by washing with distilled water. A capacitance decrease could be obtained after injection of glucose. The electrode constructed similarly but with ascorbic acid or fructose only showed a small response compared with glucose. The stability and reproducibility of the biosensor were also investigated.