High-performance biosensors were fabricated by efficiently transferring enzyme onto Pt electrode surfaces using a polydimethylsiloxane (PDMS) stamp. Polypyrrole and Nafion were coated first on the electrode surface to act as permselective films for exclusion of both anionic and cationic electrooxidizable interfering compounds. A chitosan film then was electrochemically deposited to serve as an adhesive layer for enzyme immobilization. Glucose oxidase (GOx) was selected as a model enzyme for construction of a glucose biosensor, and a mixture of GOx and bovine serum albumin was stamped onto the chitosan-coated surface and subsequently crosslinked using glutaraldehyde vapor. For the optimized fabrication process, the biosensor exhibited excellent performance characteristics including a linear range up to 2 mM with sensitivity of 29.4 ± 1.3 μA mM-1 cm-2 and detection limit of 4.3 ± 1.7 μM (S/N = 3) as well as a rapid response time of ~2 s. In comparison to those previously described, this glucose biosensor exhibits an excellent combination of high sensitivity, low detection limit, rapid response time, and good selectivity. Thus, these results support the use of PDMS stamping as an effective enzyme deposition method for electroenzymatic biosensor fabrication, which may prove especially useful for the deposition of enzyme at selected sites on microelectrode array microprobes of the kind used for neuroscience research in vivo.