A simple and low-cost electrochemical sensor based on poly(phenylalanine) and function­nalized multi-walled carbon nanotubes (F-MWCNTs) modified glassy carbon electrode (GCE) was developed for the determination of vitamin B6 (VB6). The surface morphology of modified glassy carbon electrodes was investigated with scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). The electrocatalytic activities of the bare and modified electrodes were investigated in the presence of ferri-ferrocyanide redox couple using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The exchange current density (jo = 2462 µA cm-2) and electron transfer rate constant (ko = 0.002 cm s−1) were calculated using 5 mM K3[Fe(CN)6]. The electro­chemical activity of poly(phenylalanine)/F-MWCNT/GCE towards VB6 oxidation was investigated using CV. Parameters including the number of electrons transferred (n = 2), number of protons transferred (H+ = 2), electron transfer coefficient (α = 0.51) and surface concentration of VB6 (G = 0.24 nmol cm−2) were calculated. At the optimal experimental conditions, the oxidation peak current of VB6 measured by square wave voltammetry (SWV) was found proportional to its concentration in two linear ranges of 0.5 to 20 µM and 20 to 200 µM with a low detection limit (LOD) of 0.038 µM and limit of quantification (LOQ) of 0.125 µM. Finally, the sensor was successfully used to determine VB6 in soft drink and pharmaceutical formulation samples.