We report the design and the synthesis of membrane-active peptide nanostructures, as well as their use as signal transducer in a fluorimetric assay for biologically relevant analytes. Addition of hydrophobic 21-residue peptides bearing six crown ether side chains to a solution of small unilamellar vesicles loaded with a pH-sensitive fluorophore induces a rapid fluorescence increase associated with Na<sup>+</sup>/H<sup>+</sup> transport across the bilayer membrane. To demonstrate the usefulness of these peptide nanostructures in the development of simple, rapid, and sensitive detection assays for a wide range of analytes, peptide nanostructures bearing a biotin at the N-terminal position were prepared. Addition of avidin to the assay employing these modified peptides resulted in a significant change in the time-dependent fluorescence profile. Control experiments with a non-binding proteins and saturated avidin showed that the observed changes are indeed due to specific binding of avidin to biotin modified nanostructures.