Abstract:  For the stable, reliable, fast method of sexing bovine pre-implantation embryos is still play very important role in husbandry production, the amplification experiment under the condition of the two-temperature gradient PCR was done with bovine samples such as genome, cloning embryos, embryos respectively. As a result, the stable, simple, fast method of two-temperature gradient PCR for sexing bovine pre-implantation embryos was obtained, which only took 57 minutes to identify the embryo sex. A total of 30 dairy embryos were sexed with the two-temperature gradient PCR method in the study. 15 identified embryos (11 female, 4 male) were transferred into 15 recipient cows, among them 7 were pregnant after 60 days. In the end, 5 female calves were aborted in late pregnancy, and 1 female and 1male dairy calf were born. The sexes of aborted and born calves were fully in accordance with the embryo sex predetermination with PCR method.