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In this study, we reported facile synthesis of Fe₃O₄/C composite and its application for the cost-effective and sensitive determination of tryptophan (Trp) in human serum samples. Fe₃O₄/C composites were prepared by a simple one-pot hydrothermal method followed by a mild calcination procedure, using FeCl₃∙6H₂O as Fe₃O₄ precursor, and glucose as reducing agent and carbon source simultaneously. The Fe₃O₄/C composite modified glassy carbon electrode (Fe₃O₄/C/GCE) was prepared by drop-casting method. The microstructure and morphology of Fe₃O₄/C composite was characterized by powder X-ray diffraction (XRD) and scanning electron microscopy (SEM), respectively. Due to large specific surface area and synergistic effect from Fe₃O₄ nanoparticles and carbon coating, Fe₃O₄/C composite showed excellent electrocatalytic activity toward the oxidation of Trp. As a result, the proposed Fe₃O₄/C/GCE displayed superior analytical performances toward Trp determination, with two wide detection ranges (1.0−80 μM and 80−800 μM) and a low detection limit (0.26 μM, S/N = 3). Moreover, successful detection of Trp in human serum samples further validate the practicability of the proposed sensor.