Adequate serotonin levels are pivotal to human well-being; thus, serotonin can be used as a biomarker because it regulates a wide range of physical and psychological functions. As an imbalance of serotonin is highly likely to initiate the pathogenesis of various disorders, monitoring serotonin levels in real time is in high demand for the early detection of disease. We fabricated a feld-efect transistor (FET) biosensor based on aptamer-immobilized conducting polymer nanohybrids, which showed an instantaneous response toward serotonin in solution. The mechanism of serotonin detection was based on aptamer deformation after aptamer-ligand interaction and the consequential decrease in the charge carrier density of the FET template. Docking simulations with AutoDock/Vina and PyMOL were successfully used to investigate the binding site of serotonin in the loop structure of the aptamer. The fabricated FET template showed high sensitivity toward serotonin in the range of 10 fM to 100nM, and the limit of detection (LOD) was exceptionally low at 10 fM. Moreover, the selectivity toward serotonin was confrmed by observing no signal after the injection of structural analogs, functional analogs and excess physiological biomolecules. The potential clinical application of this sensor was confrmed because it remained consistent when the bufer solution was exchanged for artifcial serum or artifcial cerebrospinal fuid (CSF). † S.G.L. and S.E.S. contributed equally to this work.