Background: This study was proposed to explore the feasibility of droplet digital PCR (ddPCR) analysis for non-invasive prenatal diagnosis of Down syndrome. Materials and Methods: The authors studied maternal plasma samples from 465 pregnant women carrying euploid and trisomy 21 (T21) fetuses. A methylation-sensitive restriction endonuclease, BstUI, was used to digest the hypomethylated holocarboxylase synthetase (HLCS) gene. The authors quantified digestion-resistant HLCS gene on chromosome 21 and fetal-specific rs6636 SNP allele on chromosome 14 by droplet digital PCR analysis. Maternal plasma DNA analysis was performed by comparing the ratio of hypermethylated HLCS to fetal-specific rs6636 SNP allele between two groups. Results: Using a rs6636 SNP allele on chromosome 14 as the reference marker, the authors analyzed 78 euploid and 28 T21 plasma samples. The ratios of the numbers of positive hypermethylated HLCS and the fetal-specific C allele in the euploid and T21 samples were significantly different ( p < 0.01, Mann–Whitney rank sum test), all but two samples with the fetal-specific C allele were correctly classified, while the ratios of the numbers of positive hypermethylated HLCS and the fetal-specific G allele in the euploid and T21 samples were significantly different (p < 0.01, Mann–Whitney rank sum test); all but one sample with the fetal-specific C allele were correctly classified. Conclusions: The study demonstrated that ddPCR approach can be applied for prenatal screening of trisomy 21.