Monodisperse Fe3O4 dots with a mean size of about 2.3 nm were successfully synthesized via a polyol-hydrolysis route without adding any dispersant. Inorganic iron nitrate was used as the metal source and triethylene glycol (TEG) was used as the polyol solvent. The Fe3O4 dots were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), selective area electron diffraction (SAED), Fourier transform infrared (FTIR) spectroscopy, N-2 adsorption-desorption, and magnetization measurements. The as-synthesized Fe3O4 dots can not only be coagulated from the polyol by ethanol and acetone, but also easily redispersed in water by ultrasonication, resulting in a clear Tyndall effect. The obtained Fe3O4 dots exhibited superparamagnetism at room temperature and the saturation magnetization is much lower than those reported in previous works. The formation mechanism of the Fe3O4 dots was proposed to be the hydrolysis of iron nitrates and subsequent dehydration and partial reduction of Fe3+ to Fe2+ at elevated temperatures in TEG.