Abstract: We established a simple methodfor the preparation of DNA template from a single oocyte or early embryo by KOH/DTT-Triton X disintegration. The PCR amplification efficiency of DNA template prepared by this method was compared with that prepared by TE-proteinase K. Single oocyte, 2-cell embryo, 8-cell embryo, morula or blastocyst were separately treated by KOH/DTT-Triton X, then the DNA template was directly used to amplify mitochondrial DNA segment by PCR. The overall PCR success rate of the 3 pairs of primers was 100% (70/70), while the overall PCR success rate of single oocyte treated by TE-proteinase K was 92.9% (65/70). Difference between the two results was significant (P<0.05), and the PCR false positive rates in both groups were 0. The designed KOH/DTT-Triton X disintegrate method was efficient to the preparation of DNA template of a single early embryo. It needed only one cycle of PCR amplification to get clear aimed DNA stripe and the efficiency was high enough to meet the need of early embryonic genetic material detection.