Phase behavior of aqueous two-phase systems (ATPS) containing cationic (SDS) and anionic (CTAB) surfactants and its application to theanine extraction was studied. Results indicated the ATPS could form under the certain SDS/CTAB molar ratio; there was a reasonable consistency between the conductivity and the formation region of ATPS, and the viscosity was higher in the formation region of ATPS. Additionally, the phase ratio increased with increase of CATB concentration, and the interfacial film between the top phase and the bottom phase was resilient. Moreover, the theanine extraction with ATPS was realized in the waste liquid of tea polyphenol production (WLTPP), and the partition coefficient of theanine decreased with increase of WLTPP concentration, whereas the extraction rate of theanine increased. The partition coefficient decreased with increasing SDS/CTAB molar ratio, and the extraction rate of theanine increased with increase of SDS/CTAB molar ratio.