MicroRNAs (miRNAs, miR) are associated with multiple cellular processes and diseases. Here, we designed fluorescent DNA probes composed of stem loop-structured DNA complementary to miRNAs and fluorophore-quencher pairs [molecular beacon (MB)] to simultaneously monitor the biogenesis of miR-206 and miR-26a, which are highly expressed during myogenic differentiation. C2C12 cells were transfected with an MB targeting miR-26a and containing a 6-FAM-BHQ1 pair (miRNA-26a MB) or an MB targeting miR-206 with a Texas Red-BHQ2 pair (miRNA-206 MB). In vitro and in vivo fluorescence analysis revealed that, only in differentiated single C2C12 cell, significantly increased fluorescence signals of miRNA-26a MB, miRNA-206 MB or simultaneous incubation of both beacons were detected due to the hybridization of miR-206 or miR-26a with their respective beacons, resulting in activation of fluorescence. Our MB-based miRNA imaging system may serve as a new imaging probe for monitoring multiple miRNAs during various cellular or disease processes associated with miRNAs.