Scanning electrochemical microscopy (SECM) has been used to create and to image thin film structures of functionalized polypyrrole, to which glucose oxidase has been covalently immobilized. In addition, the SECM was applied to optimize the quality of all steps of this microfabrication process. For this purpose, imaging of the obtained structures was performed in the conventional feedback mode of the SECM with ferrocene derivatives or [Os (bpy)(2) fpy] (bpy = 2,2’-bipyridine, fpy = 4-formylpyridine). The same working mode is also used for the enzyme-modified microstructures exploiting the ability of glucose oxidase to accept the oxidized forms of these inorganic transition metal complexes as artificial cofactors. Successive images in the absence and presence of the enzymes’s substrate glucose added to the mediator solution allowed us to distinguish between the mediator regeneration caused by a heterogeneous electron-transfer reaction at the underlying gold support and the enzyme-mediated feedback. Prospects for the development of miniaturized biosensors are discussed.