Protein isolates from album seed were prepared and subjected to HIUS treatment using a probe method. The effect of ultrasonication on physicochemical, molecular and thermal characteristics were investigated and compared with those of the control. The color characteristics (L*, a*, b* values), whiteness index, solubility, foaming capacity, and foam stability and molecular weight loss of album protein isolates (APIs) have been increased significantly (p ≤ 0.05) than the native API as a result of HIUS treatment. However, HIUS treatment reduced the denaturation temperatures (Td), enthalpies of denaturation (ΔH), thermal stability, particle size, and whiteness index. With increase in ultrasonication treatment from 5 to 25 min, the Td decreased from 84.56 to 75.90 °C and the ΔH from 44.87 to 38.75 J/g. This decrease might be related to some structural and conformational changes that had occurred in APIs due to break down of molecular bonds by sonication. The highest reduction in particle size from 245.63 μm to 134.28 μm was observed in album protein isolates treated for 25 min. Most importantly, probe sonication had imparted the structural and conformational changes in API which were confimed form the results of SDS-PAGE, surface hydrophobicity, and weight loss analyses. SDS-PAGE and weight loss showed splitting of high molecular bands into lower molecular weight bands whereas, surface hydrophobicity of HIUS treated APIs was found significantly (p ≤ 0.05) higher than that of native APIs. The changes in SDS-PAGE patten, surface hydrophobicity, and weight loss subsequently enhanced solubility along with improvement in thermal and other functional properties of album protein isolates.