As a ground-based experimental method for simulating the containerless state in space, acoustic levitation provides excellent containerless and contact-free conditions for studying droplet dynamics, including droplet evaporation and phase separation. Meanwhile, the nonlinear effects of the acoustic field, such as acoustic radiation pressure and acoustic streaming, bring novel characteristics to the droplet evaporation process and phase separation process. In this work, the evaporation and phase separation of aqueous two-phase-system (ATPS) droplet composed of polyethylene glycol (PEG) and ammonium sulfate (AMS) are investigated by a single-axis acoustic levitator through the combination of image acquisition and processing technique. During the evaporation of the ATPS droplet, the square of its equatorial diameter, $ {d}^{2} $ , decreases linearly with time, and its aspect ratio, $ \gamma $ , increases linearly with time. The PEG-AMS droplet initially in the single-phase regime can enter into the two-phase regime as the water evaporates, resulting in phase separation. The phase separation of the acoustically levitated PEG-AMS ATPS droplet includes three stages: first, a large number of PEG-rich globules form inside the ATPS droplet, and then these PEG-rich globules collide, coagulate and migrate outward, and finally a horizontal layered structure of the whole droplet comes into being. The evaporation constant, the evolution of the PEG-rich globules and the AMS-rich phase area, are analyzed for ATPS droplets with different initial aspect ratios and different initial compositions. It is concluded that the greater the initial aspect ratio and the smaller the volume fraction of the PEG-rich phase, the faster the evaporation rate of the droplet is; the greater the initial aspect ratio and the lager the volume fraction of the PEG-rich phase, the faster the phase separation is. Numerical simulations show that the acoustically levitated droplets with a large aspect ratio are subjected to greater acoustic radiation pressure on the surface, and that the corresponding sound field is more intense and the acoustic streaming is stronger, which accelerates the evaporation and phase separation of the levitated droplets. These findings contribute to deepening our understanding of the motion characteristics, evaporation dynamics and phase separation of acoustically levitated droplets, and provide a foundation for studying the containerless preparation and processing the materials under acoustic levitation.