β-Cyclodextrin-polymer-modified Fe₃O₄ microspheres were synthesized using a one-pot hydrothermal method, with a β-cyclodextrin polymer as the surfactant. Investigation of the effects of the reaction time and the amount of β-cyclodextrin polymer on the formation of functionalized microspheres suggested that the main factor affecting the microsphere size was the amount of β-cyclodextrin polymer, rather than the reaction time. The obtained β-cyclodextrin-polymer-modified Fe₃O₄ microspheres were characterized using various methods. The results indicated that the functionalized magnetic microspheres were spherical, dispersible and had high saturation magnetization. The β-cyclodextrin-polymer-modified Fe₃O₄ microspheres were used for stereoselective absorption of DL-tryptophan enantiomers. The results suggested that the functionalized magnetic microspheres absorbed more L-tryptophan and had chiral discrimination ability, and can therefore be used as a stereoselective absorbent for chiral analysis.