MicroRNAs (miRNAs) play a significant role in numerous biological processes and are implicated in a range of cancers, including breast cancer. MiRNAs have the potential to be biomarkers in clinical practice because of their distorted and unique expression, especially with regard to their presence in cancer stem cells (CSCs) that have applications in cancer diagnosis and treatment. Thus, the absolute determination of miRNA expression levels is a prerequisite for exploring their applications. Nevertheless, currently available methods may not be adequate for the detection of miRNAs in CSCs due to the inherently low population of these cells. Therefore, we combined a duplex-specific nuclease (DSN)-mediated amplification strategy with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for use in this study. We designed the substrate peptide GDKAVLGVDPFR, which contains the reporter peptide AVLGVDPFR and a tryptic cleavage site (lysine at position 3) in combination with a biotinylated DNA sequence that was complementary to a target miRNA (i.e., miR-200c). Then, this newly synthesized DNA-peptide probe was hybridized with the target miRNA. Upon the introduction of the DSN, the enzyme degraded the probe into two parts where the target miRNA was integrated and thereby triggered further cleavage. The accumulated peptide fragments were ultimately digested with trypsin to release the reporter peptide for LC-MS/MS quantification. Under these circumstances, the miRNA signal was converted and amplified into a mass response of the reporter peptide. After optimization of the parameters, including temperature, hybridization/DSN time, and the amounts of DSN and streptavidin agarose beads, we demonstrated a linear detection range between 1 fM and 200 fM for miR-200c. The detection limit obtained was 3 orders of magnitude lower than those previously reported. Finally, quantification of miR-200c in breast cancer stem cells (BCSCs) and in stem cells isolated from breast tumors was performed. We also compared these data with quantitative reverse transcription PCR (qRT-PCR) results.