The present work describes a novel approach for the determination of dopamine (DA) using carbon paste electrodes modified with sulfated β-cyclodextrin (S-β-CDCPE). The electrodes show high affinity towards DA electrochemical oxidation. DA returned a concentration detection range 5 × 10−7 M to 5 × 10−4 M and a detection limit of 1.33 × 10−7 M. DA, ascorbic acid (AA), and serotonin (5-HT) in a solution mixture can be simultaneously oxidized at significantly different potentials in the presence of S-β-CDCPE, while the unmodified CPE returned an overlapping response. In particular, S-β-CD-modified carbon paste microelectrode exhibits clear peak potential separations of 0.161, 0.101, and 0.258 V (vs. Ag/AgCl) for AA/DA, DA/5-HT, and AA/5-HT, respectively, in artificial cerebrospinal fluid (aCSF).