In this study, the multiwalled carbon nanotube‐poly (aniline) (MWCNT‐PANI) composite is successfully deposited on the glassy carbon electrode (GCE) by electropolymerization as an efficient and conventional route. The structural and chemical properties of the MWCNT‐PANI was investigated by various spectroscopic and analytical techniques, scanning electron microscopy (SEM), X‐Ray photoelectron spectroscopy (XPS) and electrochemical impedance spectroscopy (EIS). After that, the electrochemical oxidation behavior of serotonin (5‐HT) in phosphate buffer solution (pH 7.4) was examined using cyclic voltammetry (CV). Besides, the MWCNT‐PANI/GCE used for the detection of 5‐HT with linear concentration range (1.0×10−7–1.2×10−5 mol L−1), low limit of detection (3.3×10−8 mol L−1) under differential pulse voltammetric (DPV) conditions. The proposed electrochemical sensor based on MWCNT‐PANI is both time and cost effective, has good reproducibility, high selectivity as well as stability for 5‐HT determination. The developed composite electrode was used to detect 5‐HT in synthetic urine sample and satisfactory results were obtained with high recoveries.