Aqueous two-phase systems (ATPSs) have been used in the chiral resolution of various enantiomers in recent years. However, it is difficult to recycle and reuse the phase-forming components and chiral selectors in these applications. Therefore, a completely recyclable ATPS that is based on two temperature-responsive polymers, viz., poly(MAH-β-CD-co-NIPAAm-DMAEMA-BMA) (PNDB-CD) and poly(NVCL-AM) (PVAm), was employed for the chiral separation of mandelic acid (MA) enantiomers. The two polymers can be recovered by simply adjusting the temperature; meanwhile, the polymer PNDB-CD can be used as a chiral selector. The factors that affect the chiral resolution process were also investigated. A maximum separation factor (α) of 1.49 was achieved via a one-step extraction under the following conditions: PVAm concentration of 14.0 wt%, PNDB-CD concentration of 9.0 wt%, MA concentration of 5.0 mg/g, and system pH of 4.0. Moreover, the two polymers can be easily recycled after the chiral separation process. The recyclable ATPS used for enantioseparation in the current study can provide new ideas for other applications by using enantioselective liquid–liquid extraction (ELLE).