A novel amphiphilic poly(ethylene glycol)-block-poly(gamma-cholesterol-L-glutamate) (mPEGPCHLG) diblock copolymer has been synthesized. The mPEGPCHLG copolymer has good biocompatibility and low toxicity. The mPEGPCHLG copolymers could aggregate into nanoparticles with PCHLG blocks as the hydrophobic core and PEG blocks as the hydrophilic shell through emulsion solvent evaporation method. The copolymers were characterized by nuclear magnetic resonance spectroscopy, mass spectrum, Fourier transform infrared spectroscopy, and gel permeation chromatography. The particle sizes, size distributions, and zeta potentials of nanoparticles can also be determined by dynamic light scattering and transmission electron microscopy. This work provides a new and facile approach to prepare amphiphilic block copolymer nanoparticles with controllable performances. This novel copolymer may have potential applications in drug delivery and bioimaging applications.(c) 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012