In this study, a DYS14 aptamer/polyaniline–reduced graphene oxide–gold nanoparticles/gold (Apt/PANi– RGO–G*NPs/Au) electrode was fabricated to detect the Y-chromosome DYS14 DNA sequence in cffDNA in the blood plasma of pregnant women and used on real and laboratory samples with high success rate. The electrochemical properties of the prepared E-DNA biosensor were characterized by CV, SWV, XRD, and EIS. The EDNA biosensor morphological characteristics were investigated by TEM, SEM, and EDX. Phosphorothioate was used to link the aptamer to PANi–RGO–G*NPs modified gold electrode. This is due to control of the adsorption polarity and increase adsorption stability. Under optimized conditions, the linear range of the analytical technique with respect to the logarithm of the target sequence concentration was 1.0 × 10 16–1.0 × 10 8 M, the detection limit was 4.26 × 10 17 M, and the limit of quantitation was 1.422 × 10 16 M. The E-DNA biosensor displayed high selectivity and sensitivity, high efficiency, and acceptable repeatability. For fetal sex detection, 12 pregnant women from the 5th to the 15th week of gestation participated in the study. Results indicated the fabricated Apt/PANi–RGO–G*NPs/Au E-DNA biosensor to be appropriate for fetal sex determination in pregnant women between the 7th and 9th week of gestation. Notably, this method can be used as a model for the study of pathogens like bacteria and viruses.
