A biomimetic electrochemical sensor based on molecularly imprinted polymer (MIP) containing chitosan and graphene oxide was designed for direct highly sensitive and selective detection of serotonin, an important neurotransmitter, with a significant role in various body functions, in real samples such as serum, tears and saliva. The composite MIP film was immobilized in a reproducible manner onto the glassy carbon electrode (GCE) via amperometry while the optimization and complete characterization of the sensor was performed with cyclic voltammetry, electrochemical impedance spectroscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM) and X‐Ray Photoelectron Spectroscopy (XPS). The direct electrochemical detection based on the oxidation signal of the target analyte was performed by differential pulse voltammetry, the biomimetic sensor exhibiting a wide dynamic range (5 nm–10 μm), with an estimated limit of detection of 1.6 nm. The sensor demonstrated reproducibility, stability in time and reusability, excellent selectivity over other possible interferents and good recoveries in real samples, being promising for biomedical applications.