It is challenging to distinguish monozygotic (MZ) twins using traditional autosomal STR genotyping due to their nearly identical genomes. As an important kind of small non-coding RNAs, microRNAs (miRNAs) are essential regulators of gene expression and considered as excellent biomarkers due to their resistance to degradation. Moreover, droplet digital PCR (ddPCR) has emerged as a powerful technique for detecting gene mutations and pathogenic microorganisms, owing to its sensitivity and reliability. We aimed to explore the differential expression of miRNAs between MZ twins using next-sequence platform and assess the reliability of differentially expressed miRNAs by ddPCR. MiRNA sequencing (miRNA-seq) revealed nine differentially expressed miRNAs shared across five pairs of twins, including hsa-miR-3620-3p, hsa-miR-6825-5p, hsa-miR-1273h-5p, hsa-miR-200a-5p, hsa-miR-3192-5p, hsa-miR-188-5p, hsa-miR-206, hsa-miR-4796-5p, and hsa-miR-6775-3p. Subsequently, the combination of real-time quantitative PCR (qPCR) and ddPCR confirmed the ability of five of these miRNAs (hsa-miR-1273h-5p, hsa-miR-3192-5p, hsa-miR-188-5p, hsa-miR-206, and hsa-miR-6775-3p) in distinguishing monozygotic twins. Furthermore, ddPCR demonstrated superior recognition accuracy compared to qPCR. Finally, we evaluated the degradation resistance of these five miRNAs under different environmental conditions. None of the five miRNAs showed a significant decrease in expression levels after being stored at room temperature for up to 180 days or undergoing 10 freeze-thaw cycles. In summary, our study revealed the potential application of miRNAs in differentiation of MZ twins and the powerful role of ddPCR in forensic medicine.