Objective <p>Oxidative stress plays a pivotal role in the pathogenesis of Down syndrome (Trisomy 21), as chromosome 21 harbors multiple genes involved in redox homeostasis and antioxidant defense mechanisms. This study aimed to evaluate the roles of transcription factors nuclear factor erythroid 2-related factor 2 (NRF2) and nuclear factor-kappa B (NFKB), along with antioxidant enzymes cystathionine-γ-lyase (CSE) and NAD(P)H dehydrogenase [quinone] 1 (NQO1) in amniotic fluid (AF) and maternal serum (MS) as potential biomarkers for prenatal screening of Down syndrome (DS).</p> Methods <p>This prospective case-control study included singleton pregnant women undergoing amniocentesis between 16 and 24 weeks of gestation at Haseki Training and Research Hospital, Istanbul. Participants were divided into two groups: 28 pregnancies with DS confirmed by karyotype analysis (DS group) and 37 pregnancies with normal karyotype results (non-DS group). Amniotic fluid and maternal blood samples were analyzed using enzyme-linked immunosorbent assay (ELISA) kits to measure the levels of selected biomarkers.</p> Results <p>NQO1 levels were significantly higher in the DS group compared to the non-DS group in both amniotic fluid (924.84 ± 475.94 vs. 505.62 ± 358.17 ng/ml, <i>p</i> &lt; 0.001) and maternal serum (716.216 ± 242.91 vs. 394.87 ± 344.86 ng/ml, <i>p</i> &lt; 0.001). NRF2 levels were significantly lower in the DS group in both amniotic fluid (3.77 ± 4.20 vs. 6.47 ± 5.53 ng/ml, <i>p</i> = 0.029) and maternal serum (7.54 ± 5.68 vs. 14.46 ± 16.53 ng/ml, <i>p</i> = 0.022).</p> Conclusion <p>The study highlights the importance of further research to validate the use of these antioxidant enzymes and transcription factors in non-invasive prenatal testing, which may reduce the need for invasive procedures and associated complications.</p> Clinical trial number <p>Not applicable.</p>