Multifunctional magnetic nanoparticles simultaneously possessing thermo-responsive properties and chiral recognition ability show great potential in enantiomeric separation. In this study, a novel type of multifunctional magnetic Fe₃O₄ nanoparticle, decorated with smart polymer brushes consisting of poly(N-isopropylacrylamide-co-glycidyl methacrylate) chains with pendent β-cyclodextrin (β-CD) units, was fabricated as a chiral nanoselector for the thermo-sensitive selective adsorption and separation of three amino acid enantiomers. These smart polymer brushes were grafted on the surface of Fe₃O₄ nanoparticles via a combination of surface-initiated atom transfer radical polymerization and ring-opening reaction. The pendent β-CD units can serve as smart receptors for selectively recognizing enantiomeric molecules via formation of stable host–guest inclusion complexes. The thermo-sensitive poly(N-isopropylacrylamide) chains can act as microenvironmental adjustors for tuning the inclusion constants of β-CD toward enantiomeric guest molecules. The prepared multifunctional magnetic nanoparticles exhibit excellent thermo-responsive adsorption and decomplexation performances toward amino acid enantiomers. Via simply changing the operation temperature, the decomplexation of amino acid enantiomers and regeneration of the smart chiral magnetic nanoparticles can be easily achieved. Besides, the magnetic properties of the regenerated smart nanoparticles enable easy recovery under an external magnetic field for reuse. Such multifunctional magnetic nanoparticles with highly chiral recognition capability, excellent thermo-sensitive adsorption and decomplexation properties toward amino acid enantiomers, and recyclability show great potential in chiral separations.