Stem-loop hairpin DNA probes have high hybridization specificity and unique selectivity to target molecules such as DNA and small molecules. DNA-templated silver nanoclusters (DNA-AgNCs) has been widely used to detect biomolecules of interest due to the photostable, bright, and efficient methods. In this study, we measured fluorescence emission of hairpin DNA upon hybridization with cDNA and mutant cDNA (cDNA-1) or mutant cDNA containing mismatched bases in the stem region (cDNA-2). Fluorescence intensity of hairpin DNA-AgNCs in the presence of cDNA was 1.80 times higher than that of hairpin DNA-AgNCs alone, but decreased to 66% in the presence of cDNA-1 containing mismatched base corresponding to the hairpin stem region. This study demonstrated that fluorescence intensities of hairpin DNA-AgNCs were dependent on hybridization with either wild-type and mutant cDNAs.