This research investigated the physiochemical and functional characteristics of chicken liver protein isolate, which was extracted by alkali (CLPI) and ultrasound-assisted alkali (UCLPI), respectively. Protein yield of UCLPI was increased by 55.4% over that of CLPI (P < 0.05). Several amino acids were significantly higher and methionine was lower in UCLPI (P < 0.05). Ultrasound treatment reduced the particle size and prolonged the stability of the protein isolate during 7 d storage. Lower pH and a higher amount of total solubilized proteins (68.5%) were found in UCLPI by comparison with CLPI (61.8%). Surface hydrophobicity increased greatly by 63.0% compared with CLPI, but the total/reactive sulfhydryl groups and the primary structure molecular weight profile between them were not significantly different (P > 0.05). However, the enthalpy of denaturation in UCLPI decreased by 41.7%, which was related to the changes in its second structure. This resulted in better water/oil holding capacity and emulsifying properties for UCLPI. The changed physiochemical characteristics and improved functional properties of UCLPI demonstrated the potential utilization of ultrasound-assisted alkaline extraction in the food industries.