Electrochemically pre-anodized Nafion-coated glassy carbon electrodes (NCGCEs) were used for the selective determination of uric acid (UA) by square-wave voltammetry. The major interference from ascorbic acid (AA) was overcome owing to the distinct ability of the pre-anodized NCGCE to yield its best performance at around pH 5. At pH 5, AA exists in the anionic form whereas UA is in the cationic form, since their pK_a values are 4.1 and 5.4, respectively, at 25 °C. Consequently, the Nafion film repels the negatively charged AA and selective sensing of UA can be achieved. Moreover, the anodized surface has a high affinity towards UA through hydrogen bonding. Hence the combination of pre-anodization and the Nafion film modification works excellently in the determination of UA. Under optimized conditions, the detection limit for UA has been improved to 10 nM (S/N = 3). The practical analytical utility of the method is demonstrated by the measurement of UA in human urine without any preliminary treatment.