A single-shot reagentless biosensor for the amperometric determination of glucose is described. The device has been produced in a novel dual-electrode configuration, whereby the working and reference/counter electrodes are printed alongside one another in a disposable format. The biosensor is based on a cobalt phthalocyanine-containing screen-printed carbon electrode coated with cellulose acetate and glucose oxidase (GOD). The organometallic redox mediator permits the detection of GOD-evolved H2O2 at markedly reduced (2O2-permeable film directly over the sensor assembly, serving to screen out interferents and electrode-fouling agents. Voltammetry and amperometry have been used to optimize the sensor's analytical performance and SEM to investigate the efficacy of producing the chemical and biological layers.