Abstract: Cattle breeding are one of the fundamental and important economic activities. Sex determination is important for the management of fetal sex that which gender is more favorable according to the different targets. Methods of sex determination are divided into two categories: invasive and non-invasive. There are variants of candidate genes for prenatal diagnosis of fetal sex. Amelogenin genes that are on both chromosomes X and Y are significantly considered for fetal sex determination. Analysis of free fetal DNA in maternal plasma and serum as anon-invasive method to gender determination is an active background for research. In this study the proliferation of free fetal DNA of Amelogenin gene in maternal blood and bovine morula embryos by using PCR for fetal sex determination were investigated. 51 blood samples from pregnant cows and also 18 samples of 5-6 days morula were collected from ranching and Centre of Breeding in Karaj. Serum preparation and extraction of DNA for each sample was done using modified phenol-chloroform extraction procedure. This primer was selected to allow the amplification of a 467 bp single fragment of the X chromosomes in female cattle and two 467 and 341 bp fragments of the X and Y chromosomes in male cattle To evaluate the agreement between predictions obtained and results of Follow up, chi-square and coefficients of Cohen's kappa and phi were used. This method could detect accurately the sex of male and females embryos by 89%.According to the results of chi-square test, there was an significant agreement (P <0.05) between the results predicted by the PCR and the results obtained from Follow up. For the value of agreement, the criterion (P <0.05) has been shown a significant agreement between the results predicted by the PCR and the results obtained from Follow up.