We obtained epitaxial single-crystal Fe3O4(001)/MgO(001) thin films by magnetron sputtering. The high quality of the grown Fe3O4 films was confirmed by reflection high-energy electron diffraction and x-ray photoelectron spectroscopy. Atomic magnetic properties of Fe3O4(001)/MgO(001) were investigated using vibrating sample magnetometry and x-ray magnetic circular dichroism. The values of saturation magnetization and magnetic moment are 407 ± 5 emu/cm3 (3.26 ± 0.04 μB/(f.u.)) and 3.31 ± 0.15 μB/(f.u.), respectively, in the Fe3O4 film as thin as 5 nm, which are close to the bulk values. The spin polarization was directly measured using spin-resolved photoemission spectroscopy. The measured spin polarization has a maximum value of −42% ± 3%, which is comparable to the theoretical value for the (2 × 2)R45° reconstructed Fe3O4(001) surface. Furthermore, the film thickness-dependent measurements indicate that the anti-phase boundaries significantly decrease the spin polarization rather than the lattice mismatch. Our results demonstrate that epitaxial Fe3O4(001)/MgO thin films grown by magnetron sputtering have desired magnetic properties, facilitating the potential application of Fe3O4-based spintronic devices.