Cross-linked microspheres consisting of optically active helical substituted polyacetylenes are reported. For preparing the microspheres, substituted polyacetylene copolymers with pendent polymerizable CC bonds are first prepared and then used as macromonomers to copolymerize with acrylates via suspension polymerization, providing cross-linked microspheres. The helical polymer segments render the microspheres with optical activity, whereas the acrylate-based polymers afford the swelling property. CD and UV-vis spectra demonstrate the optical activity of the microspheres. The microspheres preferably adsorb (R)-(+)-1-phenylethylamine, (R)-(+)-N-benzyl-1-phenylethylamine, and Boc-D-alanine, whereas released Boc-L-alanine rather more rapidly than its enantiomer.