Betacyanins have gained increasing interest as natural food dye because of their health-promoting effects. This study investigated the betacyanins extraction from peel and flesh of red-purple pitaya using ethanol/K2HPO4-based liquid biphasic partitioning system (LBPS) by optimised concentrations of ethanol, K2HPO4, NaCl and crude. Overall, the highest values of betacyanins concentration in alcohol-rich top phase (C-t), phase volume ratio (V-r) and partition coefficient (K) of betacyanins that represented an optimum betacyanins extraction from the peel (98.080%, 1.667 and 51.097, respectively) and flesh (96.256%, 2.167 and 25.764, respectively) were achieved. Lastly, conventional solvent extractions, colour characterisation and antioxidant activity evaluation were conducted.