A novel modified carbon ionic liquid electrode is prepared as an electrochemical sensor for simultaneous determination of levodopa (l-dopa) and serotonin (5-HT). The experimental results suggest that a carbon ionic liquid electrode modified with multi-walled carbon nanotubes and cobalt hydroxide nanoparticles, and coated with Nafion (Nafion/Co(OH)2–MWCNTs/CILE), accelerates the electron transfer reactions of l-dopa and 5-HT. In addition it shows no significant interferences of uric acid and ascorbic acid as electroactive coexistent compounds with l-dopa and 5-HT in biological systems. The fabricated sensor revealed some advantages such as convenient preparation, good stability and high sensitivity toward 5-HT and l-dopa determination. The DPV data showed that the obtained anodic peak currents were linearly dependent on thel-dopa and 5-HT concentrations in the range of 0.25–225 and 0.05–75 μmol L−1, respectively. The applicability of the modified electrode was demonstrated by simultaneous determination of 5-HT and l-dopa in human serum.