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A potentiometric urea biosensor is prepared by the immobilization of urease directly onto the surface of a solid-state ammonium ion-selective electrode. The enzyme is immobilized by entrapment method onto a nonactin membrane that incorporated carboxylated polyvinylchloride. The same method of immobilization method is adopted to compare the characteristics of urea biosensors based on ammonium ion-selective electrodes with those based on pH-sensitive electrodes, using the same tin-oxide (SnO2)/indium tin-oxide glass substrate. Urea biosensors based on ammonium ion-selective electrodes respond quickly and stably to changes in urea concentrations between 0.026 and 10 mM. The slope in the linear range is around 55.56±3.15 mV/decade and the detection limit is around 5 μM. The effect of urea biosensors with different pH values is considered, and the characteristics of urea biosensors based on ammonium ion-selective electrodes are described. Additionally, the experimental results from the determination of the urea using biosensors based on pH-sensitive electrodes and ammonium ion-selective electrodes are compared and discussed.