A new enzyme electrode for the determination of urea was developed by immobilizing urease in poly(vinylferrocenium) (PVF+) matrix. A PVF+ClO4- film was coated on Pt electrode at +0.7 V by electrooxidation of poly(vinylferrocene) in metylene chloride containing 0.1 M tetrabutylammonium perchlorate (TBAP). The enzyme modified electrode PVF+E- was prepared by anion-exchange in an enzyme solution in 1.0 mM phosphate buffer at pH 7.0. FTIR spectroscopy was used to identify PVF+ClO4-, and PVF+E-. UV spectroscopy was also used to prove the enzyme immobilization. The effects of polymeric film thickness, concentration of enzyme solution, immobilization time of the enzyme, pH and temperature of the medium, concentration of the buffer solution and possible interferents on the measured potential values were investigated. The potentiometric enzyme electrode developed in this study provided linearity to urea in the 5 x 10(-5) to 1 x 10(-1) M urea concentration range. The detection limit under the optimum working conditions was determined as 5 x 10(-6) M for urea. The enzyme electrode was found to be stable for 24 days. The apparent Michaelis-Menten consant (K-M app) value and the activation energy, E-a, of this immobilized enzyme system were found to be 4.48 x 10(-5) M and 4.97 kcal mol(-1) for urea, respectively. (C) 2004 Elsevier B.V All rights reserved.