Aqueous two-phase micellar system (ATPMS), an alternative to chromatography, has been considered as a promising liquid-liquid extraction technique for biomolecule purification. To improve the specificity of ATPMSs, a nickel-chelated surfactant (TX-Ni) has been fabricated. The affinity-based ATPMS formed by Triton X-114 (TX) and TX-Ni was characterized for the purification of recombinant hexahistidine-tagged enhanced green fluorescent protein (EGFP). The stability of EGFP in the ATPMS was first confirmed. Then, the affinity binding of EGFP to TX-Ni was proved by investigation of the partitioning behavior. Thereafter, EGFP was extracted directly from cell lysate by the Ni(II)-chelated ATPMS. It was found that, more impurities were removed to the micelle-poor phase with increasing NaCl concentration, and the increase of TX-Ni gave rise to a recovery of EGFP over 90%. Finally, ethylenediaminetetraacetic acid (EDTA) was used to back-extract EGFP, presenting a total recovery yield of 83% with a purity of 70%. The results indicate that the affinity-based ATPMS is promising for the primary separation of histidine-rich proteins. (C) 2014 Elsevier B.V. All rights reserved.