Herein, a novel Pluronic F127/graphene nanosheet (PF127/GN) hybrid was prepared via an one-pot process including the simultaneous reduction of graphene oxide and assembly of PF127 and GN. The nanohybrid exhibits high water dispersibility and stability in physiological environment with the hydrophilic chains of PF127 extending to the solution while the hydrophobic segments anchoring at the surface of graphene via hydrophobic interaction. The PF127/GN nanohybrid is found to be capable of effectively encapsulating doxorubicin (DOX) with ultrahigh drug-loading efficiency (DLE; 289%, w/w) and exhibits a pH responsive drug release behavior. The superb DLE of the PF127/GN nanohybrid relies on the introduction of GN which is structurally compatible with DOX. Cellular toxicity assays performed on human breast cancer MCF-7 cells demonstrate that the PF127/GN nanohybrid displays no obvious cytotoxicity, whereas the PF127/GN-loaded DOX (PF127/GN/DOX) shows remarkable cytotoxicity to the MCF-7. Cell internalization study reveals that PF127/GN nanohybrid facilitates the transfer of DOX into MCF-7 cells, evidenced by the image of confocal laser scanning microscopy. The above results indicate the potential application of this novel nanocarrier in biomedicine. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.