Abstract: Abstract: Peanut protein, an abundant and low-cost by-product of peanut oil production industry, is rich in essential amino acids. Traditionally, this protein has been used for animal feed and fertilizer, and it is desirable to find new uses for peanut protein. The ultrasound technology, as an emerging non-thermal processing physical technology, has extensive applications in food system. To improve the functional properties and expand its application range, peanut protein isolate was treated with ultrasound and acid. A volume of 100 mL of peanut protein isolate solution (the concentration was 0.01 g/mL) was prepared and adjusted to pH value of 3.0 with 0.1 mol/L HCl. The mixtures were immediately treated using an ultrasonic cell crusher with a flat tip probe at 20 kHz at 600 W ultrasonic power for 5 min (ratio of work time to break time was 2 s : 2 s). The effects of acid, ultrasound and coupled ultrasound-acid pretreatments on the solubility, ultraviolet spectra, fluorescence spectra, secondary structure and nanostructure of peanut protein isolate were investigated. The transformation mechanism of the peanut protein aggregation state induced by ultrasound and acid was also discussed. Results of solubility showed that the ultrasound pretreatment promoted the transformation from insoluble aggregates to soluble aggregates. With the pretreatments of ultrasound and coupled ultrasound-acid, solubility of peanut protein isolate was increased by 12.9% and 15.3% respectively (P<0.05). There was no significant difference (P>0.05) in solubility between control and acid pretreatment. Acid and ultrasound pretreatments caused the increase of absorbance in ultraviolet spectra and the red shift of maximum emission wavelength in fluorescence spectra, which indicated that more phenylalanine, tryptophan, and tyrosine residues were exposed outside the polypeptide chains due to molecular unfolding and subunit dissociation. The CD spectra showed that coupled ultrasound-acid pretreatment resulted in the increase in the α-helix content by 21.9 % and random coils content by 1.8 %, while β sheet decreased by 3.6% (P<0.05). The changes in the contents of the secondary structure might have been attributed to the disruptions of the interactions between local sequences of amino acids and between different parts of the molecule. Moreover, the analyses of microstructure by atomic force microscope revealed that the particle height and size decreased significantly with the pretreatments of ultrasound, acid and combination of both. Combination of ultrasound and acid was especially effective to promote dissociation of protein. Through the investigations of solubility, ultraviolet spectra, fluorescence spectra, secondary structures and nanostructure, we could find that acid and ultrasound pretreatments destroyed hydrophobic interactions of protein molecules, induced molecular unfolding, and caused more hydrophobic groups and regions inside the molecules to be exposed to the outside. As a result, the maximum emission wavelength of fluorescence shifted and particle size decreased. Therefore, we conclude that ultrasound pretreatment under acid condition can remarkably promote subunit dissociation and unfolding of peanut protein, which will provide the theoretical basis and technological support for the preparation of functional protein obtained by subunit recombination.