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This study analyzes the selectivity of a potentiometric urea biosensor prepared by immobilizing of urease directly onto the surface of an ammonium ion-selective electrode. In this investigation, using the entrapment method, the urease enzyme was immobilized onto the carboxylated polyvinylchloride (PVC-COOH) membrane surface of an ion-selective electrode containing nonactin as an ionophore and a tin oxide (SnO2) thin film. To describe the characteristics of the selectivity of urea biosensors based on ammonium ion-selective electrodes, the determinations of potentiometric selectivity coefficients (Kpoti,j) of the urea biosensor in different univalent interfering ions were presented in a sample solution of pH 7.5. Using the separate solution method and fixed interference method (FIM) for determining selectivity coefficients, this investigation considers and discusses the influence of the urea biosensors preference on the primary ions relative to interfering ions of the same charge number. The observation results indicate that FIM is the proper method for calculating the selectivity coefficients of the ammonium ISE-based urea biosensors.