A novel nonionic microemulsion electrokinetic chromatography (MEEKC) method has been developed and validated for the determination of uric acid. The optimum microemulsion consisted of 80 mmol L⁻¹ Brij-35, 2% (v/v) n-heptane, 14% (v/v) 1-butanol, 2.5% (v/v) acetonitrile and 10 mmol L⁻¹ sodium borate buffer (pH 9.25). Under the optimum conditions, the method had a good linear relationship (correlation coefficient: 0.9996) with a range of 5–200 μg mL⁻¹ for uric acid. The limit of detection for uric acid was 0.50 μg mL⁻¹. The intra-day RSD values for migration time and peak area ratio were less than 0.71% and 0.78% (n = 5), and inter-day values were less than 1.64% and 1.82% (n = 5), separately. The method was applied for the quantitation of uric acid in gout patients' plasma and urine with recoveries in the range of 99.84–119.36% and 97.15–108.48%, respectively.