In this work, a novel enantioselective MCE was developed employing BSA-conjugated Fe3O4@Au nanoparticles (Fe3O4@Au NPs) as stationary phase. Fe3O4@Au NPs with high magnetic responsively, excellent solubility, and high dispersibility in water were prepared through a sonochemical synthesis strategy. BSA was then immobilized onto the Fe3O4@Au NPs surfaces through the well-developed interaction between Au NPs and amine groups of BSA to form Fe3O4@Au NPs-BSA conjugates, which were then locally packed into PDMS microchannels with the help of magnets. The resultant Fe3O4@Au NPs-BSA conjugates not only have the magnetism of Fe3O4 NPs that make them easily manipulated by an external magnetic field, but also have the larger surface and excellent biocompatibility of Au shell, which can incorporate much more biomolecules and well maintain their biological activity. In addition, the successful BSA decorations endowed Fe3O4@Au NPs-BSA conjugates with pH-tunable water solubility related to the pI of BSA (pI 4.7) and led to enhanced stability against high ionic strength. Compared with the native PDMS microchannel, the modified surfaces exhibited more stable and suppressed electroosmotic mobility, and less nonspecific adsorption toward analytes. Successful separation of chiral amino acids (tryptophan and threonine) and ofloxacin enantiomers demonstrate that the constructed MCE columns own ideal enantioselectivity. The results are expected to open up a new possibility for high-throughput screening of enantiomers with protein targets as well as a new application of magnetic NPs.