A chemically modified electrode based on the carbon nanotube film-coated glassy carbon electrode (GCE) is described for the simultaneous determination of dopamine (DA) and serotonin (5-HT). The multiwall carbon nanotube (MWNT) film-coated GCE exhibits a marked enhancement effect on the current response of DA and 5-HT and lowers oxidation overpotentials. The responses of DA and 5-HT merge into a large peak at a bare GCE, but they yield two well-defined oxidation peaks at the MWNT film-coated GCE. The experimental parameters were optimized, and a direct electrochemical method for the simultaneous determination of DA and 5-HT was proposed. The interference of ascorbic acid (AA) was investigated, and the results showed that a large excess of AA did not interfere with the voltammetric responses of DA and 5-HT. The modified electrode has been successfully applied for the assay of 5-HT and DA in human blood serum.