A glucose amperometric biosensor based on glucose oxidase immobilized on an overoxidized polypyrrole (PPyox) platinum modified electrode, by glutaraldehyde co-crosslinking with bovine serum albumine, is described. The advantages of covalent immobilization techniques (e.g. high loading and long-term stability of the enzyme) are coupled with the excellent interferent rejection of electrosynthesized non-conducting polymers. The sensor showed an apparent Michaelis-Menten constant of 16 +/- 0.8 mM, a maximum current density of 490 microA/cm2 and a shelf lifetime of at least 3 months. Ascorbate, urate, cysteine and acetaminophen at their maximum physiological concentrations produced a glucose bias in the low micromolar range. Flow-injection response was linear up to 20 mM glucose with typical sensitivity of 84.0 +/- 1.5 nA/mM. The sensor was tested for glucose determination of untreated serum samples from both normal and diabetic subjects; results of amperometric assay compared well with those obtained by a standard enzymatic-colorimetric method.