A novel glassy carbon electrode (GCE) modified with bromothymol blue (BTB) and multi-walled carbon nanotubes (MWNTs) is described, and the electrochemical behavior of 5-fluorouracil (5-Fu) on the modified electrode was investigated by cyclic voltammetry. Various kinetic parameters such as transfer electron number and diffusion coefficient were calculated. A good linear relationship between the oxidation peak current and logarithmic concentration of 5-Fu in range of 8 × 10−7 to 5 × 10−3 M (R2 = 0.9981) was obtained in phosphate buffer solution (pH 6.8); the detection limit was 2.67 × 10−7 M (S/N = 3) by use of MWNTs/BTB/GCE. It was successfully applied to the 5-Fu determination in injections.