Millets have emerged as a healthy dietary substitute to cereals due to their nutritional and functional attributes and therapeutic potentialities due to the generation of bioactive peptides from their native protein. The aim of the study is to explore the structural, functional and thermal properties of isolated millet protein using ultrasonic wave treatment and exploring it as an alternative protein for food applications. Pearl millet protein (PMP) was isolated using acid-base precipitation of defatted flour, and ultrasound (US) treatment (at different interval: 5, 10 and 15 min with 45KHz) was performed to improve the yield and functional properties. In US-assisted PMP samples, the particle size (789.30 – 1321 nm) and zeta potential (-15.20 to -23.96 mV) altered due to changes in the structure of the protein matrix. The FTIR spectra of control and US-assisted PMP samples also shows a significant difference, which may due chemical breakdown and changes in the secondary structure. Circular dichroism spectra also show that the various protein fractions (i.e. ɑ- and β- helix, turn, and random) have undergone significant modifications. The distinct XRD peaks in 8 - 19⁰ revealed the presence of α-helical and β sheets structures and the tiny or diffused peaks result of processing used in protein isolation. The SEM images of PMPs revealed that US-assisted PMP samples appears more uniform size in comparison to the unsonicated ones, with a larger, compact protein structure that broke down into disordered and irregular fragments. Thermal analysis provided insights into the thermal degradation behavior of the protein, while Nuclear Magnetic Resonance spectroscopy confirmed the presence of amino acids and organic acids or their derivatives. The intervention of US in millet protein extraction could be beneficial as it induces several functional changes to protein that ultimately impacted its solubility and practical application in food.