Abnormal urea levels are important clinical indicators of kidney and metabolic disorders. Therefore, developing sensitive and reliable urea detection systems is crucial for clinical diagnostics as well as applications in food quality control and environmental monitoring. In this study, a chitosan (CS)-stabilized hybrid nanocomposite composed of a copper-based metal–organic framework (Cu-MOF) and single-walled carbon nanotubes (SWCNTs) was synthesized and used to construct an enzymatic electrochemical biosensor. Urease was immobilized onto the CS-SWCNTs/Cu-MOF-modified glassy carbon electrode to enable selective urea recognition. The sensor design parameters, including the optimal pH, temperature, and concentrations of the nanocomposite and enzyme, were systematically optimized to enhance analytical performance. The resulting biosensor demonstrates excellent sensing performance in detecting urea, showing a wide detection range of 0.1–20 mM, a sensitivity of 2.65 μA.mM−1.cm−1, and a low detection limit of 1.3 µM. It also provides a rapid response time of 5 s, good operational stability, satisfactory reproducibility, and a storage lifetime of up to 30 days. Furthermore, the sensor demonstrates strong selectivity against common interferents and achieves accurate urea quantification in human serum samples, highlighting its potential for clinical use.
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