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Serotonin (5-HT) is a critical neurotransmitter involved in many neuronal functions, and 5-HT depletion has been linked to several mental diseases. The fast release and clearance of serotonin in the extracellular space, low analyte concentrations, and a multitude of interfering species make the detection of serotonin challenging. This work presents an electrochemical aptamer-based biosensing platform that can monitor 5-HT continuously with high sensitivity and selectivity. Our electrochemical sensor showed a response time of approximately 1 min to a step change in the serotonin concentration in continuous monitoring using a single-frequency EIS (electrochemical impedance spectroscopy) technique. The developed sensing platform was able to detect 5-HT in the range of 25–150 nM in the continuous sample fluid flow with a detection limit (LOD) of 5.6 nM. The electrochemical sensor showed promising selectivity against other species with similar chemical structures and redox potentials, including dopamine (DA), norepinephrine (NE), L-tryptophan (L-TP), 5-hydroxyindoleacetic acid (5-HIAA), and 5-hydroxytryptophan (5-HTP). The proposed sensing platform is able to achieve high selectivity in the nanomolar range continuously in real-time, demonstrating the potential for monitoring serotonin from neurons in organ-on-a-chip or brain-on-a-chip-based platforms.