This article focused on the assessment of the potential of Raman spectroscopy for the determination of structural changes in black-bean protein isolate (BBPI) dispersions with low-frequency (20 kHz) ultrasonication applied at various powers (150, 300 or 450 W) and for different durations (12 or 24 min). It also reported on differential scanning calorimetry analyses. A decrease in TD at low- and medium-power ultrasonication confirmed these ultrasonication treatment disrupted internal hydrophobic interactions of protein molecules and broke up unstable aggregates to smaller soluble protein aggregates, while an increase in TD at high-power was attributed to repolymerization of aggregates. Raman spectroscopy analysis revealed a decrease in the α-helix proportion and an increase in β-sheets after ultrasonic treatment except Sample E (300 W, 24 min). Transformation of aggregation results in a reconstruction in secondary structure of BBPI, especially in β-sheet structure. Ultrasonic-treatment induced a decrease in the normalized intensity of the Raman band near 760 cm-1 which indicated that Tryptophan residues tended to expose and also indicated protein partially unfolding. No significant difference was found in Tyr doublet ratios between unheated and ultrasound-treated BBPI indicated that ultrasound did not change the microenvironment around tyrosyl residues. While the intensity of 1 450 cm-1 band increased with increasing ultrasonic intensity and treatment time, and then decreased with further increase in power and treatment time. In general, the formation of aggregation transferred g-g-t conformation to t-g-t conformation. Though some mechanism of aggregation-repolymerization of BBPI remains to be clearly defined, Raman spectroscopy provide a feasible tool to study the structural changes of BBPI prepared under different ultrasonic conditions, give a new perspective to elucidation of protein structure.