The voltammetric oxidation and determination of 5-Fluorouracil 5-(FU) was studied at a glassy carbon electrode (GCE) in the presence of cetyltrimethyl ammonium bromide (CTAB) by cyclic and differential pulse voltammetry at pH-7. The results indicated that the voltammetric responses of 5-Flurouracil are drastically increased in the low concentration of CTAB, suggesting that CTAB exhibits observable enhancement effect to the determination of 5- Flurouracil. Under the optimal conditions the peak current was proportional to 5-Flurouracil concentration in the range of 2.0 x 10-8 to 6.0 x 10-7 M with detection limit of 20.13nM by differential pulse voltammetry. The proposed method was applied to the determination of 5- Fluorouracil in pharmaceuticals. The analytical performance of this sensor has been evaluated for detection of analyte in human serum and urine as real samples.