In this study, one aqueous two-phase system (ATPS) composed of polymer and salt was employed to extract sulfated polysaccharides (sulfated dextran) to polymer-rich phase from model polysaccharides mixture (dextran and sulfated dextran), and another ATPS was employed to back-extract sulfated polysaccharides to a salt phase. This process was further applied to separate sulfated Enteromorpha polysaccharides (EPs) from crude EPs. For optimal PEG1000/(NH4)(2)SO4 (TLL45) ATPS, the extraction efficiencies were 93.2 +/- 0.1%, 95.4 +/- 2.0%, 56.3 +/- 4.4% for 5, 50, 500 kDa sulfated dextran in top PEG-rich phase and were 91.9 +/- 1.4%, 97.1 +/- 2.9%, 95.2 +/- 0.9% for 5, 50, 500 kDa dextran in bottom salt-rich phase, respectively. The 500 kDa sulfated dextran presented precipitation at the interface, and the extraction efficiency was 26.9 +/- 5.7% for 500 kDa sulfated dextran in the interface. Different ATPSs composed of PEG1000 and another salt (sodium citrate, K2HPO4, sodium tartrate, etc.) were performed to back-extract sulfated polysaccharides of PEG-rich phase to another salt phase. Under the PEG1000/sodium tartrate ATPS, the best back-extraction efficiencies of 5, 50, 500 kDa sulfated dextran were 75.1 +/- 4.1%, 86.1 +/- 0.2% and 88.3 +/- 1.6%, respectively. Crude EPs was extracted by ultrasonic/microwave-assisted extraction from Enteromorpha powder with yield of 9.5% (w/w, dry algae powder). After that, crude EPs was separated by ATPSs, and analyzed using anion exchange chromatography and FT-IR. The results of anion exchange chromatography indicated that crude EPs contained three fractions (EP-1, EP-2, EP-3). The results of crude EPs separating by ATPSs demonstrated that majority of EP-3 concentrated in top PEG rich phase, and EP-2 in bottom salt-rich phase of PEG1000/(NH4)(2)SO4 ATPS. FT-IR results showed that EP-3 presented S=O and C-O-S strength vibrations at 1254 and 848 cm(-1) (SO42- group), while EP-2 had no correlative strength vibrations. These results revealed that an ATPS method was established preliminarily for sulfated polysaccharides separation from a natural polysaccharides mixture.