In this paper, two novel amino acid chiral ILs, tetramethylammonium-l-arginine (TMA-l-Arg) and tetramethylammonium-l-aspartic acid (TMA-l-Asp), were applied for the first time in CE enantioseparation to evaluate their potential synergistic effect with glycogen as chiral selector. As observed, significantly improved separation of tested enantiomers were obtained in the chiral ILs/glycogen synergistic systems compared to the single glycogen separation system. Several primary parameters affecting the enantioseparation, such as amino acid ILs (AAILs) concentration, glycogen concentration and buffer pH, were systematically investigated. An achiral tetramethylammonium hydroxide ionic liquid (TMA-OH) modified separation system was also evaluated to validate the superiority of the novel chiral ILs/glycogen synergistic systems. To further optimize the overall synergistic systems, the effect of three other parameters, including buffer concentration, applied voltage and capillary temperature were simultaneously analyzed by a central composite design (CCD), and excellent enantioseparations were achieved with the optimized parameters. The results indicate that the application of chiral ILs/glycogen synergistic systems is a promising way in chiral separation science.