Two pairs of nested primers were derived from the 5′ flanking sequence of the bovine SRY gene to serve as male-specific sexing primers, which were used for PCR-based sex determination of bovine embryos in combination with casein protein gene primers as internal control. At the same time, methods of DNA extraction from bovine embryo cells were established and the sensitivity of nested PCR and normal PCR was studied for developing a technique of sex determination of bovine embryos adapted for practical use. Results showed that the primers used in the study were male-specific and all primers were bovine-specific. Boiling and freeze–thawing were used for DNA extraction from embryo cells. Amplification products were obtained with only 10 cells by nested PCR, so this system was used for the sex determination of bovine embryos. The sex of 10 Holstein cow embryos was identified and the results were confirmed upon birth of the calves.