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Synthesis of magnetite (Fe3O4) nanoparticles (NPs) with high purity, stability, and small sizes for biomedical applications is in high demand. The research on Fe3O4 NPs has been rapidly expanding and holds considerable promise for advancing new technologies and enhancing existing ones. Herein, Fe3O4 was synthesized from iron ferrous chloride (II) and sodium hydroxide (NaOH) on air, and then coated with gum Arabic (GA) in a single step. The characterizations were investigated using UV–Vis absorption, ultra-high-resolution scanning electron microscope (SEM), Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), and vibrating sample magnetometer (VSM). Compared to bare Fe3O4, GA-coated magnetite nanoparticles (GA-Fe3O4) showed a uniform cubic morphology with a mean size of 8.61 ± 1.31 nm. Stability studies were performed via dynamic light scattering (DLS) technique, zeta (ζ) potential, and observing the precipitation of NPs over 5 weeks. GA-Fe3O4 showed higher stability of -34.5 mV compared to -23.1 mV for bare Fe3O4. Further, GA-Fe3O4 NPs demonstrate interesting magnetic properties that can be efficiently separated from the reaction medium with the aid of an external magnet. The magnetic properties of GA-Fe3O4 were confirmed through VSM analysis, revealing a significantly high saturation magnetism of approximately 226 emu/g. GA-Fe3O4 NPs are promising magnetic NPS that can offer several benefits in biomedical applications, such as increased biocompatibility, improved stability, and enhanced functionality. [ABSTRACT FROM AUTHOR]