Iron (III) oxide nanoparticle (Fe3O4NP) was synthesized via green route using Callistemon viminalis leaf extract. A composite of Fe3O4NP with functionalized multi-walled carbon nanotube (f-MWCNT) was then applied as glassy carbon electrode modifier to develop a choline sensor. Electrocatalytic oxidation of choline on nanocomposite modified electrode (GCE/f-MWCNT/Fe3O4NP) was studied by cyclic voltammetry while mechanism of electron transfer process during choline electrocatalytic oxidation was monitored by electrochemical impedance spectroscopy at fixed potential 0.5 V with frequencies between 100 kHz and 0.01 Hz. Active surface coverage area of choline, charge transfer coefficient and number of electron transferred were reported. Square wave voltammetry (SWV) was employed for the determination of choline over a linear concentration range of 24.3 - 215.6 μM. A sensitivity of 0.0244 μA/μM and 9.9 μM detection limit was found. The sensor was selective to choline (0.1 mM) in the presence of 100 mM and 0.1 mM ascorbic acid (AA) and dopamine (DA) respectively using SWV and chronoamperometry. The designed electrode was reproducible and stable. The sensor was successfully used for choline detection in choline dietary supplements with resultant good recoveries. The fabrication of the sensor was simple and cost-effective. Figure 1