Creatinine is an important biomarker of kidney function and muscular metabolism. In this paper, we developed the β-lead dioxide/single-walled carbon nanotube (β-PbO/CNT) and the β-PbO/CNT ion-selective electrode (β-PbO/CNT/ISE), which were used as highly sensitive potentiometric sensors for creatinine detection. The fabricated electrodes exhibited highly pH-sensitive characteristics due to the synergistic effect of the electrochemical properties of CNT and β-PbO. Moreover, an ammonium-ion-selective membrane coating allowed the β-PbO/CNT electrode to be NH-selective for direct detection of the ammonium ion. By exploiting the electrochemical characteristics of these electrodes, the creatinine assay was established through the one-step selective conversion of creatinine by creatinine deiminase, in which the OH and NH generated by the enzymatic reaction were detected using β-PbO/CNT and β-PbO/CNT/ISE electrodes as pH- and NH-responsive sensors, respectively. The total creatinine assay can be completed within ∼5 min. The assay results from β-PbO/CNT and β-PbO/CNT/ISE showed excellent sensitivity values of -75.56 and 64.62 mV in the detection range of 10-400 μM with a fast response (20 s), and the limits of detection were calculated to be 0.06 and 0.13 μM, respectively. Moreover, the developed creatinine sensor showed high selectivity against 11 interfering bio/chemical species with negligible interferences (selectivity coefficient <10) and excellent repeatability (>97% within 25 cycles) and long-term stability for 4 weeks of storage. In addition, the feasibility and practicality of the device were successfully demonstrated in human serum tests, with recoveries of 95-104% for PbO/CNT and 92-110% for PbO/CNT/ISE.