Digital in-line holographic microscopy (DIHM) has attracted attention because of its simple but powerful three-dimensional (3D) imaging capability. To improve the spatial resolution, 3D image reconstruction algorithms use numerical magnification, which generates distortions in the generated images. We propose a method to overcome this problem by using the simple relation between the object and image positions in 3D space. Several holograms were taken while translating a resolution target at different axial positions by a motorized stage. We demonstrated the effectiveness of our method by reconstructing the 3D positions of 3-μm-diameter polymer beads on a tilted slide glass from a single measured hologram.