Modular allosteric aptamers with discrete recognition and signaling regions provide a facile method of carrying out label-free detection by forgoing complex target labeling requirements. Herein, we describe the design and function of an aptamer scaffold capable of forming a hairpin loop in the presence of FAD (the signaling trigger). The aptamer includes a recognition region for the microRNA (miR) Let-7i. Upon selective miR hybridization, the aptamer undergoes a conformational shift to release FAD and thus produce a measurable response. As a result, the described method can sensitively and selectively detect miR Let-7i with a wide linear range of 0.1 pM to 1 μM and a detection limit of 150 fM. Additionally, this strategy was able to selectively discriminate between sequences with 1- and 2-nucleotide (nt) differences.