Magnetic tunnel junctions using an epitaxial magnetite (Fe₃O₄) layer were prepared on Si/SiO₂, MgO {100}{110}, and {111} single-crystal substrates, and their properties were studied. Tunnel junctions of U.L./Fe₃O₄/Al-oxide/CoFe were fabricated by rf sputtering. An epitaxial Fe₃O₄ layer was grown by sputtering and was found to be highly oriented in it’s crystal axis in the film plane. Each Fe₃O₄ showed markedly different MR properties depending on the epitaxy planes of the MgO single-crystal substrate. The Fe₃O₄/Al-oxide/CoFe film deposited on {110} MgO showed the highest MR ratio of 10% when the field was applied along the easy axis of the epitaxial Fe₃O₄. The MR properties of the film deposited on {110} MgO exhibited a strong angular dependence in the film plane.