Abstract A major concern of medicine today is the sustained release of therapeutic compounds. Delivery vehicles for such compounds must be biocompatible. Ideally, loading a drug into the delivery vehicle will be a simple process, and vehicle properties will allow control over the drug release profile under desired conditions. Here, polypeptide multilayer nanofilms have been prepared by electrostatic layer-by-layer self-assembly to study the post-fabrication loading and release of a model therapeutic, methylene blue (MB). Drug loading and release have been characterized by optical spectroscopy for different peptide designs at different pH values, and film surface morphology has been characterized by atomic force microscopy (AFM). Differences in peptide structure have been found to influence MB loading and release under otherwise fixed conditions. Release is also influenced by pH, salt concentration, and number of “capping” layers. Although more research will be needed to exhaust the potential of polypeptide multilayer films, present results would suggest that the technology holds considerable promise for applications in medicine.