Atomic force microscopy (AFM) is a powerful technique for imaging molecules, macromolecular complexes, and nanoparticles with nanometer-resolution. However, AFM images are distorted by the shape of the tip used. These distortions can be corrected if the tip shape can be determined by scanning a sample with features sharper than the tip and higher than the object of interest. Here we present a 3D DNA origami structure as fiducial for tip reconstruction and image correction. Our fiducial is stable under a broad range of conditions and has sharp steps at different heights that enable reliable tip reconstruction from as few as ten fiducials. The DNA origami is readily co-deposited with biological and non-biological samples, achieves higher precision for the tip apex than polycrystalline samples, and dramatically improves the accuracy of the lateral dimensions determined from the images. Our fiducial thus enables accurate and precise AFM imaging for a broad range of applications.