Cell-free fetal DNA (cffDNA) from fetal calves was isolated in the bovine maternal plasma in the fifth week of pregnancy, accounting for around 10% of the total. Recently, cffDNA has been used as a non-invasive prenatal screening technique for aneuploidy, genetic disorders in humans, early pregnancy diagnosis, and animal fetal sex determination. This study's objective was to identify the presence of cffDNA in the blood of 13 dairy cows (11 pregnant, 2 non-pregnant, and 1 unmarred) gathered from farms in Can Tho, the Mekong Delta, Vietnam. Plasma / Serum cfc-DNA Purification Midi Kit (Product # 55600) was used to separate cffDNA. Two PCR-designed primers named SRY-137 and GAPDH-109 were employed to amplify the Y-chromosome-specific sequences SRY and the housekeeping gene GAPDH, respectively. The fetal sex was indicated by PCR data corresponding to the calf's sex at birth in 11 cases. The study's findings first proved the existence of cell-free fetal DNA circulating in the plasma of a pregnant cow in Vietnam and established the calf's sex using simple, high-precision procedures. The use of cffDNA for sex screening brings up the possibility of quickly determining the gender and screening for genetic diseases in a big herd of cattle and animals with long gestation periods.