Skipjack tuna (Katsuwonus pelamis) liver (TL) contains high-quality proteins which can potentially serve as an excellent source of functional protein ingredients. Thus, this study was conceptualized to evaluate the physicochemical, functional, and biological properties of proteins from TL using the pH shift process. The pH shift process was conducted through solubilization of TL at pH from 1.5 to 12.5, and the solubilized proteins at pH 2.5, 3.5, 10.5 and 11.5 were precipitated at pH 5.5. Finally, the tuna liver protein powders after the processes at pH 2.5 and 11.5 (TLP 2.5 and TLP 11.5, respectively) were obtained by freeze-drying, i.e. those with the highest extraction and protein recovery yields under acidic and alkaline conditions. Protein and lipid contents of TLPs were higher and lower, respectively, compared to the TL powder (control). Glutamic acid, aspartic acid, and alanine were prominent amino acids found in both TLPs. Foaming properties and water/oil holding capacity were higher in TLP 11.5, while protein solubility and emulsion properties were greater in TLP 2.5 compared between groups. Additionally, the DPPH • and ABTS •+ scavenging activities, as well as the angiotensin I-converting enzyme inhibitory activity, were remarkably higher in TLP 11.5 than in TLP 2.5. On the other hand, significant ferrous-ion chelating activity was observed in TLP 2.5. In conclusion, TLP 11.5 could serve as an alternative functional protein ingredient that provides essential amino acids, functional properties, and bioactivities.