Mesoporous glass beads and silica were modified with α-cyclodextrin (CD) for the separation of xylene isomers by means of direct bonding, linking spacer group, and cross-linking agent. The separation efficiency of the α-CD modified silica particles was tested by using simple column chromatography. A strong inclusion complex formation of p-xylene was observed from the column packed with α-CD bonded phases. Spacer group and crosslinking agent decreased the formation of the inclusion complex of p-xylene and increased selectivity between m-xylene and o-xylene. The inclusion complex formation of p-xylene was strongly diminished when the mobile phase was switched from methyl alcohol to toluene. The column packed with the cross-linked α-CD bonded silica showed the best performance in the separation of xylene isomers.