A composite picture of multi-wall carbon nanotubes (MWCNTs) with Nafion (NF) cation exchange membrane is prepared by cast deposition on glassy carbon electrodes. A sensitive electrochemical sensor for the detection of bilirubin (BR) at the NF/MWCNTs hybrid modified glassy carbon electrode was offered. The electrochemical behaviors of bilirubin at the NF/MWCNTs hybrid electrode were studied in acetone containing sulfuric acid as the supporting electrolyte. Compared with bare glassy carbon electrode (GCE), the proposed integrated sensor, improved analytical performance characteristics in the electrochemical oxidation of bilirubin. The electrochemistry of bilirubin on NF/MWCNTs hybrid electrodes were studied by electrochemical impedance spectroscopy, cyclic voltammetry (CV) and square-wave voltammetry (SWV). The oxidation peak current of bilirubin obtained at the NF/MWCNTs modified GC electrode was 10 times higher than that of the unmodified glassy carbon electrode. To optimize the experimental conditions, a linear calibration plot for bilirubin was obtained between 0.8 mu M and 10 mu M with the detection limit of 0.014 mu M. Finally, the proposed sensor was successfully employed to determine bilirubin in human urine samples.