The voltammetric separation of dopamine and ascorbic acid was studied with cyclic voltammetry at two kinds of carbon nanotube-modified electrodes (coated and intercalated). The anodic peak difference reached 270 mV under the present conditions. The separation mechanism and effect factors were carefully studied. Using various types of surfactants as coating dispersants of carbon nanotubes, it was demonstrated that the charge nature of the surfactants had a strong effect on the electrochemical behavior of dopamine and ascorbic acid. When the oxidation solution of carbon nanotubes was changed from the most commonly used mixed concentrated nitric acid and sulfuric acid (1 + 3 v/v) to dilute nitric acid and to hydrochloric acid, the anodic peak separation value of dopamine and ascorbic acid increased significantly, and it was shown that carboxylic acid groups attached to the carbon nanotubes were an adverse factor for the discrimination of DA from AA. These results indicated that the resolution of DA and AA was mainly attributable to the stereo porous interfacial layer formed from aggregated pores and inner cavities of the carbon nanotubes. The modified electrodes exhibited an attractive ability to measure DA and AA simultaneously and showed good stability and reproducibility.