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This work develops and validates an electrochemical approach for uric acid (UA) determinations in both endogenous (cell lysate) and physiological (serum) samples. This approach is based on the electrocatalytic reduction of enzymatically generated H2O2 at the biosensor of uricase−thionine−single-walled carbon nanotube/glassy carbon (UOx−Th−SWNTs/GC) with the use of Th−SWNTs nanostructure as a mediator and an enzyme immobilization matrix. The biosensor, which was fabricated by immobilizing UOx on the surface of Th−SWNTs, exhibited a rapid response (ca. 2 s), a low detection limit (0.5 ± 0.05 μM), a wide linear range (2 μM to 2 mM), high sensitivity (90 μA mM−1 cm−2), as well as good stability and repeatability. In addition, the common interfering species, such as ascorbic acid, 3,4-dihydroxyphenylacetic acid, 4-acetamidophenol, etc., did not cause any interference due to the use of a low operating potential (−400 mV vs saturated calomel electrode). Therefore, this work has demonstrated a simple and effective sensing platform for selective detection of UA in the physiological levels. In particular, the developed approach could be very important and useful to determine the relative role of endogenous and physiological UA in various conditions such as hypertension and cardiovascular disease.