This study aimed to verify the reliability of the 7 tissue differentially methylated regions used in the methylated DNA immunoprecipitation (MeDIP) real-time quantitative polymerase chain reaction (real-time qPCR) based approach of fetal DNA in maternal blood to diagnosis of fetal trisomy 21. Forty pregnant women with high risk pregnancy who were referred after first or second trimester screening tests, were selected randomly. For each sample whole DNA extraction (mother and fetus), fragmentation of DNA, immunoprecipitation of methylated DNA and real- time qPCR using 7 primer pairs, was performed. D-value for each sample was calculated using the following formula D= -4.908+ 0.254 X_EP1+ 0.409 X_EP4+ 0.793 X_EP5+ 0.324 X_EP6+ 0.505 X_EP7+ 0.508 X_EP9+ 0.691 X_EP12. In all normal cases, D value was negative, while it was positive in all trisomy cases. Therefore, all normal and trisomy 21 cases were classified correctly which correspond to 100% specificity and 100% sensitivity for this method. The MeDIP real-time qPCR method has provided the opportunity for noninvasive prenatal diagnosis of fetal trisomy 21 to be potentially employed into the routine practice of diagnostic laboratories.