With the development of 3D integrated packaging technology, failure analysis is facing more and more challenges. Defect localization in a 3D package is a key step of failure analysis. The complex structure and materials of 3D package devices demand non-destructive defect localization technology for full packages. Magnetic field imaging and three-dimensional X-ray technology are not affected by package material or form. They are effective methods to realize defect localization on 3D packages. In this paper, magnetic field imaging and high-resolution three-dimensional X-ray microscopy were used to localize the open defect in a 3D package with a TSV daisy chain. A two-probe RF method in magnetic field imaging was performed to resolve isolation of the defect difficulties resulting from many different branches of TSV daisy chains. Additionally, a linear decay method was used to target sub-micron resolution at a long working distance. Multiple partition scans from a high-resolution 3D X-ray microscopy with a two-stage magnification structure were used to achieve sub-micron resolution. The open location identified by magnetic field imaging was consistent with that identified by a three-dimensional X-ray microscope. The opening was located on the top metal in the proximity of the fifth via. Physical failure analysis revealed the presence of a crack in the top metal at the opening location.