We report on the preparation and structural properties of MgO/Fe bilayers (a MgO thin film on an Fe thin film), Fe/MgO bilayers (an Fe thin film on a MgO thin film), and Fe/MgO/Fe trilayers, all grown by magnetron sputtering on SrTiO3(001) substrates. We investigated the structural properties of these heterostructures with X-ray diffraction (XRD) (θ–2 θ scan, rocking curve and pole figure measurements), atomic force microscopy (AFM), and high-resolution transmission electron microscopy (HRTEM). Single-crystalline epitaxial growth was confirmed by the X-ray pole figure measurements for both the MgO/Fe and Fe/MgO bilayers, which were the basic layer components for the Fe/MgO/Fe trilayer. The cross-sectional HRTEM clearly indicated epitaxial growth of the Fe/MgO/Fe trilayer structures on the SrTiO3(001) substrates with excellent flat interfaces having roughness of less than a few angstroms.