Human hair, composed primarily of keratin, represents a sustainable waste material suitable for various applications. Synthesizing keratin nanoparticles (KNPs) from human hair for biomedical uses is particularly attractive due to their biocompatibility. In this study, keratin was extracted from human hair using concentrated sulfuric acid as the hydrolysis agent for the first time. This process yielded KNPs in both the supernatant (KNPs-S) and precipitate (KNPs-P) phases. Characterization involved scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), Zeta potential analysis, X-ray diffraction (XRD), and thermogravimetric analysis (TG). KNPs-S and KNPs-P exhibited average diameters of 72 ± 5 nm and 27 ± 5 nm, respectively. The hydrolysis process induced a structural rearrangement favoring β-sheet structures over α-helices in the KNPs. These nanoparticles demonstrated negative Zeta potentials across the pH spectrum. KNPs-S showed higher cytotoxicity (CC = 176.67 µg/mL) and hemolytic activity, likely due to their smaller size compared to KNPs-P (CC = 246.21 µg/mL), particularly at concentrations of 500 and 1000 µg/mL. In contrast, KNPs-P did not exhibit hemolytic activity within the tested concentration range of 32.5 to 1000 µg/mL. Both KNPs demonstrated cytocompatibility with fibroblast cells in a dose-dependent manner. Compared to other methods reported in the literature and despite requiring careful washing and neutralization steps, sulfuric acid hydrolysis proved effective, rapid, and feasible for producing cytocompatible KNPs (biomaterials) in single-step synthesis.