Lyophilized wool nanoparticle powders (L-WNPP) derived from wool fibers, were extracted through the neutralization method. Here, we propose the mechanism of wool nanoparticle (WNP) formation in an aqueous solution. The effects of pH, alkali concentration, and alkali treatment time on the size and morphology of WNP were investigated. Results confirmed that WNP were well dispersed and stable in the aqueous solution and globular particles were identified with an average size of 50 nm by using 3% sodium hydroxide (NaOH) after a 4-h treatment through dynamic light scattering (DLS), zeta potential, scanning electron microscopy (SEM), and transmission electron microscope (TEM) analyses. Furthermore, fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analyses showed that the L-WNPP maintained the chemical structure of wool protein. X-ray photoelectron spectroscopy (XPS) analysis demonstrated that higher concentrations alkaline would destroy most amide bonds of molecule chains. Moreover, L-WNPP were further used to prepare transparent protein nanoparticle films. Our results will provide guidelines for developing protein nanoparticles for biotechnology fields that require size-controlled nanoparticles. (C) 2019 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved.