Cyclodextrin (CD) based chiral stationary phases (CSPs) have simulated great attention due to their unique chiral recognition ability. Functionalized cyclodextrins bonded silica gel as chiral stationary phases have been dramatically developed due to their chemical stability and solvent tolerability. To explore the functionalization of phenylcarbamoylated β-cyclodextrin CSPs on their enantioselectivities, 4-chloro-3-methylaniline and 5-chloro-2-methyl phenyl isocyanate were employed. Two CSPs have been thus developed by clicking the CD derivatives onto alkynylated silica support. They include per-4-chloro-3-methylphenylcarbamoylated β-cyclodextrin clicked CSP (CSP1) and per-5-chloro-2-methylphenylcarbamoylated β-cyclodextrin clicked CSP (CSP2), which have both electron-donating (methyl) and withdrawing (chlorine) groups in the phenylcarbamate moieties, but different locations. The CSPs were successfully characterized in terms of structure using nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR) and elementary analysis. Their enantioselectivities were evaluated in reversed-phase high performance liquid chromatography (HPLC). The comparison study on the enantioseparation of nine flavonoids with the two CSPs demonstrates the higher enantioselectivities of CSP1 over CSP2, because of the different locations of electron-donating (methyl) and withdrawing (chlorine) groups in the phenylcarbamate moieties of CD derivatives. The baseline enantioseparations achieved in water/methanol as mobile phase offered great potential for the CSPs to be used in practical application.