Downstreaming vaccine production faces a challenge in adopting numerous chromatography and filtration steps to efficiently purify large viral particles. Purity, potency, and quality are the main problems that plague virus separation and purification. This article aims to evaluate a three-stage PEG-Salt aqueous two-phase extraction (ATPE) system to purify foot-and-mouth disease virus (FMDV). Unique separation efficiencies were obtained by selecting phase-forming salt and varying the molecular weight (MW) of PEG and the pH value by using neutral salt additives. Improving the purity of FMDV and removing the phase-forming polymer were performed by multi-stage ATPE system. Results revealed that the partition coefficient (K) of FMDV in single-stage ATPE did not linearly increase with the MW of PEG. The highest K value and recovery of FMDV were 98.9% and 107.69, respectively, when the concentrations of PEG 6000 and sodium citrate were 14% and 16%, respectively. With the use of the three-stage APTE, the purity of FMDV increased from 0.007 to 0.14, PEG was eliminated, and the overall recovery of FMDV reached 72%. The results of the 1000 g scale-up experiment are consistent with those of the 10 g system without centrifugation.