The invention discloses a method and a system for X ray detection on multilayer package substrate defects. The method comprises the following steps of 1, projection data acquisition, 2, two-dimensional cross-sectional image reconstruction, 3, three-dimensional image reconstruction, 4, three-dimensional image virtual-cutting, 5, SURF feature-based registration on a two-dimensional slice X and the corresponding standard slice image, 6, corresponding matching point existence determination on the standard slice image according to a transformation matrix H, 7, opening operation on an obtained two-dimensional image, 8, connected domain labeling, 9, defect position computation and defect gray level value information determination, and 10, substrate defect mechanism determination. The method utilizes an X ray three-dimensional imaging technology to acquire cross-sectional images of the multilayer package substrate and to detect physical defects such as through holes and interconnection between the substrates, realizes automatic determination on the substrate defect mechanism by expert system evaluation on the various physical defect data, and improves production technology reliability.