Down Syndrome (DS) is the most common chromosomal abnormality characterized by neurodevelopmental impairment. Apart from maternal age, its risk factors remain poorly understood. This prospective case-control study aimed to evaluate the role of maternal anti-zona pellucida (ZP) antibodies (Ab) and anti-thyroid-Ab in predicting DS. Correlations of anti-ZP-Ab and anti-thyroid-Ab with maternal age were also assessed. Anti-ZP-Ab were measured after childbirth using ELISA. Anti-thyroid peroxidase (aTPO) and anti-thyroglobulin (aTgII) antibodies were also analysed with the Allelica IM platform. Statistical analyses included receiver operating characteristic curve assessment, expressed as area under the curve (AUC) and linear regression modeling. Between September 2020 and October 2022, 58 women were enrolled. Anti-ZP-Ab levels were significantly higher in women with DS pregnancy with an odds ratio adjusted for maternal age of 71.52 (95% CI: 7.05–725.18) and an excellent predictive performance (AUC = 0.94; 95% CI: 0.88–1.00). For optical density levels > 1, the accuracy was 89.7% (95% CI: 78.2–100.0). No statistically significant differences were observed for aTPO and aTgII. Neither Anti-ZP-Ab nor anti-thyroid antibodies increased with age. These findings suggest that Anti-ZP-Ab are strongly associated with DS risk, suggesting a potential age-independent autoimmune contribution to trisomy 21. Their evaluation may support preconception counseling, especially for women aged > 35 years. Future studies could clarify causality and define the role of maternal autoimmunity in DS etiology.