In this work, three macrocyclic beta-cyclodextrin (beta-CD) derivatives, namely carboxymethyl-beta-cyclodextrin (CM-beta-CD), sulfobutyl ether-beta-cyclodextrin (SBE-beta-CD), and hydroxypropyl-beta-cyclodextrin (HP-beta-CD), were employed in the formation of novel aqueous two-phase system (ATPS) with several hydrophilic solvents respectively. It was observed that anionic beta-CD derivatives (CM-beta-CD and SBE-beta-CD) facilitated the ATPS formation and the obtained binodal curves showed a decreasing order in inducing ATPS as acetonitrile approximate to 1-propanol > 2-propanol > ethanol. Furthermore, the proposed ATPS as a novel enantio-selective liquid-liquid extraction technique was evaluated by enantioseparating racemic zopiclone (ZPC). Opposite partitioning orientations for ZPC in two groups of the investigated ATPS were studied systematically and 1-propanol/CM-beta-CD ATPS was selected as the optimal enantioseparation system. Under the optimal conditions, moderate enantioseparation efficiency (alpha=2.58 and e.e.%=32.66%) and high recovery (R-T(S)=95.54% and R-T(R)=98.22%) were achieved simultaneously via a single run of extraction. (C) 2015 Elsevier Ltd. All rights reserved.