The development of a new urea biosensor is described by immobilizing urease in poly(vinylferrocenium) (PVF+) matrix. A PVF+ClO4 film was coated on Pt electrode at +0.7 V versus an Ag/AgCl by electrooxidation of poly(vinylferrocene) in methylene chloride containing 0.1 M tetrabutylammonium perchlorate (TBAP). The enzyme modified electrode PVF+E- was prepared by anion-exchange in an enzyme solution in 50 mM phosphate buffer at pH 7.0. 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 were investigated. The amperometric enzyme electrode developed in this study provided linearity to urea in the 1-250 mu M urea concentration range. The detection limit under the optimum working conditions was determined as 1 mu M for urea. The enzyme electrode was found to be stable for 29 days. The apparent Michaelis-Menten constant (K-M app) value and the activation energy, Ea, of this immobilized enzyme system were found to be 2.54 x 10(-4) M and 5.90 kcal mol(-1) for urea, respectively. (c) 2005 Elsevier B.V. All rights reserved.