Here, we report on a ratiometric fluorescence biosensor based on self-assembled peptide nanostructures (SPN), which can respond to conformational changes induced by RNA ligand binding. The design of the SPN biosensor was inspired by the conformational stabilization and multimerization behaviors of the HIV-1 Rev protein induced by cooperative protein protein and protein RNA interactions. Because conformation-sensitive SPN biosensors can orchestrate binding and signal transduction events, they can be developed as highly sophisticated and smart nanomaterials for biosensing.