The invention discloses a preparation method of a printed circuit board failure region sample. The method comprises the following steps of slice sampling: through electric performance test, determining a sampling region of a printed circuit board sample, making a mark, and cutting the sample in the required slice dimension; uniformly mixing epoxy resin glue liquid, a curing agent and a catalyst; then, pouring the mixture into a special metallographic slice mold in which the slice sample is placed; performing still standing curing; after the curing completion, taking out the sample to be ground and polished from the mold; grinding and polishing: monitoring the distance from the surface of a region to be tested and the surface of the sample to be ground and polished, wherein the distance height is the grinding thickness; stopping grinding after the grinding is performed to the preset thickness; polishing; obtaining the printed circuit board failure region sample. The preparation method has the advantages that a slice sampler with high automation degree is used for precise sampling; the detecting efficiency and the accuracy and reliability of the detecting result are improved.