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A biosensor is proposed for selective and sensitive detection of glucose. This electrochemical amperometric bienzymatic glucose biosensor is constructed by co-immobilization of horseradish peroxidase and glucose oxidase on vertically aligned carbon nanofibers (VACNFs). An enzyme wiring technique is used to plug the enzymes with the metal electrode using VACNFs, which facilitates an effective way to transfer electrons from the electrode to the electrochemical reaction centers. Direct electron transfer of horseradish peroxidase at the VACNF electrode is observed. Mediator or membrane free operation of this biosensor can potentially result in the application of these sensors in environmental monitoring, healthcare, as well as in varieties of scientific experiments. Experimental results indicate that the proposed biosensor can detect very low level of glucose (as low as 0.4 μM). Operational characteristics of the bienzymatic biosensor in terms of detection limit, sensitivity, and selectivity are also examined.