Rapid and simple micellar electrokinetic chromatography (MEKC) with in-capillary derivatization and fluorescence detection has been developed to determine morphine in human serum. The sample was introduced into a background electrolyte (BGE) containing potassium ferricyanide, whereas morphine was oxidized into highly fluorescent product, pseudomorphine. Different parameters for derivatization and subsequent separation were systematically investigated for the analysis of morphine in serum. Efficient performance of the developed MEKC system was carried out in a single run using BGE made up of 70 mM sodium tetraborate decahydrate (pH 10.5), 0.30 mM potassium ferrricyanide, 80 mM sodium dodecyl sulfate, and applied voltage of 9 kV. The combination of MEKC with in-capillary derivatization of morphine was successfully achieved with a high degree of sensitivity. The validation of the method showed good linearity between areas of morphine and the corresponding concentrations over the range of 5-5000 ng/mL. Excellent accuracy and precision were obtained at all concentration levels. The mean recoveries of morphine were ranging from 83.86 to 94.45%. The validated MEKC method successfully permitted determination of morphine in clinical samples after a single oral dose of controlled release morphine sulfate tablets.