In this paper, a new stable and sensitive electrochemical sensor was prepared by two-fold modification of glassy carbon electrode (GCE) with poly-alizarin red S (AzrS) and multiwalled carbon nanotubes (MWCNTs). The fabricated poly-AzrS/MWCNTs/GCE modified chemical sensor was employed towards the investigation of electrocatalytic oxidation of serotonin in 0. 1 mol/dm3 phosphate buffer (PBS) solution of pH 6. 0 with the help of voltammetric techniques such as cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). The developed electrochemical sensor exhibited a potent and persistent electron mediating behaviour towards the serotonin determination and also lavishly resolved oxidation peaks for serotonin and epinephrine (EP) simultaneously. The effect of experimental variables, such as potential scan rate, solution pH, simultaneous determination, accumulation time, impedance and concentration were examined. The limit of detection (LOD) and limit of quantification (LOQ) were found to be as 1. 8×10-7 mol/dm3 and 17. 52×10-7 mol/dm3, respectively with a dynamic range from 0. 5×10-6 to 1. 1×10-5 mol/dm3. The fabricated electrochemical sensor was applied for the direct determination of serotonin in blood serum samples. Further the fabricated two-fold modified electrochemical sensor exhibited good sensitivity, stability and reproducibility in comparison with bare GCE towards the determination of serotonin.