This paper describes a novel polymeric matrix, poly (p-phenylenediamine), as glucose oxidase entrapment media for amperometric glucose determination. The glucose sensor was constructed in a one-step procedure by anodic electropolymerization of p-phenylenediamine and concomitant incorporation of glucose oxidase at a potential of 0.7 V versus Ag/AgCl. Results show that the new glucose sensor has a linear range up to 6 mM, fast response time (less than 4 s) and high sensitivity. The specificity of the enzyme electrode for possible interfering species such as lactose, sucrose and urea was tested, and no discernible anodic signal was detected above the background current. Moreover, the effects of pH and temperature on the amperometric response were systematically investigated and the activation energy for enzymatic reaction was calculated as 41 kJ/mol. Also, the biosensor was found to be stable for more than 30 days.