Although serotonin (5-HT) is one of the most important neurotransmitters that are responsible for critical roles in the central nervous system, only few studies have been reported on the reliable and sensitive detection of 5-HT by the SERS technique owing to the lower Raman cross-section and lack of a common extraction method of 5-HT. In this study, a SERS-active substrate was fabricated by electrodepositing hierarchical Au nanostructures on amine-terminated ITO (indium tin oxide) glass to achieve an enhanced Raman signal of 5-HT. The electrodeposition process was performed by applying the nucleation potential E1 = 0.7 V for 2 s and the growth potential E2 = -0.2 V for 1000 s for repeated cycles. As a testing analyte, 5-HT was dissolved in four different solvents i.e. PBS buffer, Tris-HCl buffer, distilled water, and ethanol solution (20%). Among them, Tris-HCl buffer produced the most sensitive and reproducible Raman signals for 5-HT along with the lowest detection limit of ∼10-9 M due to the strong adsorption interactions between the Au surface and the indole amine groups of 5-HT under alkaline conditions. Surprisingly, the standard curve between the Raman intensity at 751 cm-1 and the log-scale concentration of 5-HT showed a linear dynamic range of over five orders of magnitude in the Tris-HCl buffer solution, suggesting a possible quantification of the 5-HT level in a biological fluid with 5-HT dysfunction or deficiency.