This study aims to determine the effects of various alkaline pHs on the nutritional and functional properties of protein isolated from defatted chia flour (DCF). The DCF isolated using alkali extraction method at pH 8.5, 10.0, and 12.0 were coded as CPI-8.5, CPI-10.0, and CPI-12.0, respectively. The highest extraction yield and protein recovery yield was demonstrated by CPI-12.0 (19.10 and 59.63%, respectively), with a total protein content of 74.53%, and glutelin showed the highest portion (79.95%). The CPI-12.0 also demonstrated the most elevated essential (36.87%), hydrophobic (33.81%), and aromatic (15.54%) amino acid content among other samples. The DCF exhibited the highest water (23.90 gg) and oil (8.23 gg) absorption capacity, whereas the CPI-8.5 showed the highest protein solubility (72.31%) at pH 11. DCF demonstrated the highest emulsifying capacity at pH 11 (82.13%), but the highest stability was shown at pH 5 (82.05%). Furthermore, CPI-12.0 at pH 11 shows the highest foaming capacity (83.16%) and stability (83.10%). Despite that, the CPI-10.0 manifested the highest antioxidant capacity (DPPH: 42.48%; ABTS: 66.23%; FRAP: 0.19), as well as ACE-I (35.67%). Overall, the extraction pH had significant effects in producing chia protein isolates (CPI) with improved nutritional and functional qualities.