Cell-free fetal RNA has been highlighted as useful tools for the fetal sex determination or other genetic inherentdisorder. However, there is no knowledge about the sex determination using cell free fetal RNA in bovine field. Thus,the present study aimed to evaluate the presence of transcripts of DDX3Y, USP9Y and ZRSR2Y genes in maternalplasma of pregnant cows to determine the sex of the fetus using real-time quantitative polymerase chain reaction assay,and verify its accuracy, sensitivity and specificity compared with the molecular testing and the calf sex at birth. Transcripts of USP9Y and DDX3Y genes were expressed in the all plasma of males and females both the control groupand the experimental group. However, ZRSR2Y gene was matched up with the molecular testing and the true sex incontrol group and has an overall accuracy of 82.6%, a sensitivity of 75%, and a specificity of 100% in experimentalgroup. Therefore, these results indicated that real time PCR technique, as a noninvasive and cost-efficient method, ispossible to determination fetal sex in the bovine species using circulating cell free RNA in maternal plasma and especiallyZRSR2Y gene could be a good candidate for the RNA based sex determination work.