Immobilization of NAD-dependent redox enzymes on electrode surfaces has been widely reported for the construction of enzymatic biosensors for detection of various biomolecules in different industries. Molecular wiring of such redox enzymes facilitate an efficient electron transfer by connecting redox center of the enzyme with the electrode surface. Iron-sulfur complexes are known for being the firstlink between protein and the mediating molecules in the mitochondrial electron transport chain to abstract electrons from NAD⁺. The application of iron-sulfur molecules as molecular wires to mediate electron transport between NAD-dependent glucose dehydrogenase (NAD-GDH) and gold electrode surface (i.e. a glucose bioanode) was studied. Methodology for fabrication and characterization of iron-sulfur based glucose biosensor at different construction stages were described. The biosensor performance characteritics of the novel Fe-S based glucose biosensor was investigated using amperometric analyses in the presence of glucose.