Amylase, which can hydrolyze starch into oligosaccharides, is a significant enzyme with diverse applications in various industries. In comparison to microbial amylases, plants can serve as a rich source of plant-derived amylase for industrial production at lower cost and toxicity. In this study, the ethanol/phosphate aqueous two phase system (ATPS) was adopted to recover the amylase from the red pitaya peel. The effects of several ATPS parameters such as concentrations of ethanol and phosphate, the system pH, and the concentration of sodium chloride (NaCl) on the recovery of amylase were investigated. Based on these results, a central composite design (CCD) was used to optimize the recovery of amylase from the red pitaya peel. Results demonstrated that the optimal conditions for recovery of amylase were observed for the ATPS containing 19% (w/w) ethanol, 26% (w/w) phosphate pH 7.0, and 3.8% (w/w) NaCl. The amylases were recovered to the ethanol-rich top phase with an optimum yield and a purification factor of 92.48% and 5.75, respectively. Therefore, this study demonstrated the feasibility of ethanol/phosphate ATPS as a promising tool for the simultaneous recovery and purification of amylase from the red pitaya peel.