In this study, magnetite (Fe3O4) nanoparticles were electrochemically synthesized in an aqueous electrolyte at a given potential of -1.3 V for 180 s. Scanning electron microscopy revealed that dendrite-like Fe3O4 nanoparticles with a mean size of < 80 nm were electrodeposited on a glassy carbon electrode (GCE). The Fe3O4/GCE was utilized for sensing chloramphenicol (CAP) by cyclic voltammetry and square wave voltammetry. A reduction peak of CAP at the Fe3O4/GCE was observed at 0.62 V, whereas the uncoated GCE exhibited a very small response compared to that of the Fe3O4/GCE. The electrocatalytic ability of Fe3O4 was mainly attributed to the formation of Fe(VI) during the anodic scan, and its reduction to Fe(III) on the cathodic scan facilitated the sensing of CAP. The effects of pH and scan rate were measured to determine the optimum conditions at which the Fe3O4/GCE exhibited the highest sensitivity with a lower detection limit. The reduction current for CAP was proportional to its concentration under optimized conditions in a range of 0.09-47 μM with a correlation coefficient of 0.9919 and a limit of detection of 0.09 μM (S/N=3). Moreover, the fabricated sensor exhibited anti-interference ability towards 4-nitrophenol, thiamphenicol, and 4-nitrobenzamide. The developed electrochemical sensor is a cost effective, reliable, and straightforward approach for the electrochemical determination of CAP in real time applications.