Polyvinylferrocene (PVF) was electrochemically deposited on platinum and carbon electrodes to form a stable and resilient film. During cyclic voltammetry in phosphate buffer, the PVF film deposited on carbon electrodes exhibited anodic and cathodic peaks at 214 and 68 mV, respectively. Both types of electrodes, bearing electrodeposited PVF and crosslinked glucose oxidase, were responsive to glucose, but the carbon electrode appeared to provide a faster response and could determine glucose between 0.1 and 8 mM. When protected by a layer of polymer electrochemically formed from resorcinol and phenylenediamine, the mediated biosensors based on PVF-deposited carbon electrodes were capable of determining glucose up to 25 mM with a response time of 1 min, for at least 50 repeated analyses with good reproducibility. The presence of ambient oxygen, ascorbic acid (0.1 mM), and uric acid (0.5 mM) did not affect their performance. When applied for the determination of the glucose level in reconstituted human serum, the results agreed well with those of the reference hexokinase assay.