A novel method for biphasic extraction and separation of different polysaccharides from Radix Sophorae Tonkinensis (RST) was developed by microwave-assisted aqueous two-phase extraction (MAATPE) in a one-step procedure. Using ethanol/Na2HPO4 system as a green extractant, the effects of the composition of the aqueous two-phase system (ATPS), extraction temperature and extraction time were investigated by single-factor experiments coupled to response surface methodology (RSM) through UV-vis analysis. Under the optimum conditions, extraction yields of two polysaccharides from top and bottom phases were 8.52 +/- 0.21% (w/w) and 2.18 +/- 0.08% (w/w), respectively. Compared with heating solvent extraction (HSE) and ultrasonic assisted extraction (UAE), MAATPE exhibited higher extraction efficiency. By means of Fourier transform infrared spectroscopy (FT-IR) and high-performance gel permeation chromatography (HPGPC), two polysaccharides obtained from both phases had the characteristics of homogenous heteropolysaccharides, but there were some differences in chemical structures and the molecular weight from each other. Both polysaccharides could be extracted respectively from RST to the top ethanol-rich phase and the bottom salt-rich phase due to their polarity difference. Furthermore, the chemical compositions of two polysaccharides were confirmed by high-performance liquid chromatography (HPLC) analysis after hydrolysis and derivatization, the molar ratios of monosaccharides were glucose: arabinose: galactose: rhamnose: mannose: glucuronic acid: aminogalactose = 63.70: 18.21: 7.00: 5.10: 3.13: 1.78: 1.07 for the polysaccharide in the top phase and 75.24: 3.79: 3.71: 3.37: 2.51: 1.43: 9.95 for those in the bottom phase, respectively. By means of extraction process analysis and scanning electron microscopy (SEM), the exportation of MAATPE mechanism revealed that combining microwave-assisted extraction (MAE) with aqueous two-phase extraction (ATPE) not only improved the extraction yields, but also obtained at least two different polysaccharides. Under microwave field, the ATPS could make the herb cell ruptured, and multiphase mass-transfer was accelerated. The proposed method is a rapid, simple and efficient alternative to biphasic extraction of diverse polysaccharides from natural plants.