We report the design and synthesis of enzyme-responsive nanofibers. The fibers are composed of self-assembled hydrophobic beta-sheet peptides incorporating protease-sensitive domains, fluorescent reporters, and hydrophilic poly(ethylene glycol) (PEG) units. Using urokinase plasminogen activator (uPA) as a model system, nanofibers were developed to release fluorescent fragments upon uPA incubation. These protease-sensitive nanofibers may have considerable biomedical applications as diagnostic sensors or for protease-assisted drug deliveries.