Introduction <p>Down syndrome, caused by the triplication of human chromosome 21 (trisomy 21 [T21]) or its distal segment, is the most prevalent chromosomal anomaly associated with intellectual disability in newborns.</p> Objectives <p>To investigate the relationship between T21 and hydrops by analyzing metabolomic alterations, identifying correlations, and exploring pathway regulation mechanisms. This study goes beyond biomarker discovery, aiming to elucidate the pathogenesis of T21 and to explore the underlying mechanism.</p> Methods <p>We developed a ¹²C₂-/¹³C₂-isotope dansylation labeling LC-MS workflow to profile amine/phenol-based metabolomic differences in amniotic fluid (AF) between T21 and euploid fetuses.</p> Results <p>This workflow enabled the classification of AF specimens, revealing 138 increased and 116 decreased out of 2351 detected metabolites in T21 AF specimens. Metabolite identities were confirmed via LC-MS/MS spectral analysis using authentic standards. Dysregulated metabolites in T21 AF included markers of oxidative regulation and glutathione metabolism as well as those linked to fetal development. Further subgroup analysis identified 31 T21-associated metabolites, including significantly elevated androsterone sulfate in T21 AF both with and without hydrops. Among 30 hydrops-associated metabolites, most were reduced in hydrops AF, while hyaluronic acid (HA) was notably elevated only in T21 hydrops cases. Correlation analyses highlighted negative associations between HA and metabolites like kynurenine and homovanillic acid, suggesting potential roles in immune modulation and neuronal development in the fetal microenvironment.</p> Conclusion <p>This study identifies T21-associated metabolites that may serve as early diagnostic markers or therapeutic targets, offering insights into the metabolic landscape of Down syndrome and a foundation for exploring fetal therapeutic strategies.</p>