Polyethyleneglycol-modified surfaces have become popular for anti-biofouling applications. In this paper, a novel glucose biosensor was fabricated by immobilizing glucose oxidase (GOx) onto the carboxymethyl-PEG-carboxymethyl (CM-PEG-CM) biomaterial film on the surface of glassy carbon electrode (GCE). The biosensor was applied in whole blood directly, which was based on the low values of polymer-water interfacial energy, resistance to protein adsorption and cell adhesion. The entrapped GOx could preserve its bioactivity and exhibited an excellent electrochemical behavior with a formal potential of -290 mV in phosphate buffer solution (PBS) (pH=7.4). Response studies to glucose were carried out using differential pulsed voltammetry (DPV). The results indicated that the modified electrode can be used to determine glucose without interference from L-ascorbic acid (AA) and uric acid (UA) with the low detection limit of 1.24 x 10(-5) M. The data obtained from the biosensor showed good agreement with those from a biochemical analyzer in hospital. The GOx biosensor modified with CM-PEG-CM will have essential meaning and practical application in future that attributed to the effect of anti-biofouling and good performance. (C) 2012 Elsevier Ltd. All rights reserved.