Zinc oxide–potassium ferricyanide (ZnO–KFCN) composite film was prepared on ITO coated corning glass using pulsed laser deposition (PLD). The composite film has proved to be a suitable platform for enzyme immobilization. The composite matrix exhibits the advantages of ZnO along with enhanced redox property due to the presence of a mediator in the matrix. Glucose oxidase (GOx) has been chosen as the model enzyme for studying the application of the present matrix to biosensing. The sensing response of the bio-electrode, GOx/ZnO–KFCN/ITO/glass, towards glucose was studied by electrochemical and photometric techniques. The bio-electrode exhibits good linearity and low value of Michaelis–Menten constant. Due to efficient biosensing in a mediator-less system the present bio-electrode should lead to a hand held integrated lab-on-chip device.