C/Fe nanocomposite (CFN) was synthesized by a procedure similar to an exfoliation/adsorption process to intercalate Fe(3+) into graphite oxide (GO) layers and would be reduced in a H(2) atmosphere. The results of X-ray diffractometry (XRD) and transimission electron microscopy (TEM) show that the form of CFN is carbon nanotube-Fe nanoparticle composite with alpha-Fe distributed on the nanotube wall. Paste electrode has been constructed using CFN mixed with paraffin. The electrochemical characteristics of such carbon-Fe nanocomposite paste electrode (CFNPE) has been compared with that of carbon paste electrode (CPE) and evaluated with respect to the electrochemistry of potassium ferricyanide, ascorbic acid and cysteine by cyclic voltammetry. CFNPE can accelerate the electron-transfer to improve the electrochemical reaction reversibility. To fabricate the third-generation glucose biosensor, glucose oxidase (GOD) was immobilized on CFNPE surface with Nafion covered after a pretreatment. Oxygen, ascorbic acid and uric acid have no interference with the glucose detection. The biosensor displays a remarkable sensitivity and stability and the results used in the determination of glucose in the human serum samples are satisfactory.