Abstract: A highly sensitive sensor for the amperometric assay of glucose in aqueous media having a face-to-face sandwich configuration is provided which comprises a sensing electrode strip containing a first redox mediator in an electrically conductive coating and a reference electrode strip in simultaneous contact with a reagent strip containing a second redox mediator which is sandwiched between the active electrode surfaces. An opening is provided in the reference electrode for the introduction of samples. In one embodiment, a whole blood separation membrane is interposed between the reference electrode and the reagent strip to filter red blood cells and other particles from whole blood to enable direct assay of glucose without sample preparation. The sensing electrode comprises a non-conductive support member having an electrically conductive layer containing the first redox mediator.