Chicken slaughterhouses can produce extensive waste, mainly feathers, that needs to be managed properly to prevent environmental pollution. Low cost, biodegradability, low immunogenicity, and colloidal stability of recycled feather keratin makes it attractive to produce protein-based nanomaterials, which is the objective of this study. Based on feather keratin's inherent ability to self-aggregate, we hypothesize that it can be used to form small nanoparticles to create nanostructures in physiological media for potential medical purposes. Here, urea and 2-mercaptoethanol (2 ME) were used to solubilize feather keratin without damaging its primary structure. Fluorescence, Fourier transform infrared (FTIR), and circular dichroism (CD) spectroscopies revealed that, after removing urea and 2 ME by dialysis, keratin molecules were able to form new intra-molecular disulfide and hydrogen bonds, along with hydrophobic interactions to regain their three-dimensional conformation. They further self-aggregated into extremely small uniform 13.33 nm diameter nanoparticles, measured by field emission scanning electron microscopy (FESEM), with 22.83 nm hydrodynamic diameter, measured by dynamic light scattering (DLS). The FTIR and CD spectroscopy showed that the beta-sheet content of the keratin molecules increased after nanoparticle formation, and the Congo Red test indicated that they were partially amyloid in nature. The nanoparticles had a high tendency to establish interparticle bonds, leading to new arrangements in the form of nanofibrils, nanolayers, and larger nanoparticles. In a novel approach, the type of buffer, incubation time, temperature, pH, and concentration of keratin molecules were adjusted to control the formation of one-, two-, and three-dimensional nanostructures. The self-assembly behavior of small nanoparticles under non-degradative solubilization and controlled pH, temperature, and concentration, was utilized, for the first time, to generate nano-architected structures with predictable characteristics. The results provide a platform for the development of new biomedical keratin nanomaterials/nanocomposites on a large scale.