An enzymeless biosensor, based on electrode position of overoxidized polypyrrole nanofiber onto pencil graphite electrode and modified with cobalt(II) phthalocyanine tetrasulfonate (CoPcTS), was investigated in this study. CoPcTS showed electrocatalytic activity for the oxidation of glucose in alkaline solution. The electrochemical performance of the modified electrodes was investigated by differential pulse voltammetric (DPV) method. The resulting biosensor exhibited excellent performance for glucose determination with a wide linear range (0.25-20 mM), a highly reproducible response (R.S.D. of 2.7%), low percentage of the interferences and long-term stability. The calculated detection limit was 0.1 mM at 3 sigma. in order to verify the reliability of the biosensor, it was applied to the determination of glucose in serum samples. The results were satisfactory and agreed closely with those measured in a hospital. (C) 2008 Elsevier B.V. All rights reserved.