A novel, easy and accurate capillary electrophoresis (CE) coupled with flow injection (FI) method for the separation and determination of aspartic acid (Asp) enantiomers by on-line derivatization had been developed, and it had been applied to the real sample for the first time. The derivatization reagents were o-phthalaldehyde (OPA) and mercaptoethanol (ME), which were obtained easily, the chiral selector was beta-cyclodextrin (beta-CD), the micellar chemical was sodium dodecyl sulfate (SDS), and the modifier was methanol. By on-line derivatization, aspartic acid enantiomers were automatically and reproducibly converted to the ultraviolet (UV)-absorbing diastereoisomer derivates, which were separated by micellar electrokinetic chromatography (MEKC). According to the factors affecting the separation and sensitivity of aspartic acid enantiomer and other amino acids in the real sample, the pH value and concentration of the buffer, the concentration of beta-CD and SDS, the volume percentage of the methanol (v/v) in the buffer, the applied voltage and the conversion time were selected as the investigating variates. Under the investigated separation conditions, D-aspartic acid (D-Asp), L-aspartic acid (L-Asp) and other four amino acids achieved the baseline separation in not only the standard mixture of amino acids but also the real sample (Compound Amino Acid Injection (6AA)). The repeatability (defined as relative standard deviation (RSD), n = 5) was 4.0% and 4.0% with peak area evaluation, and 4.2% and 3.7% with peak height evaluation for D-Asp and L-Asp in the real sample. Recovery at added standard levels of 1.0, 3.0 and 6.0 mM was 92%, 104% and 109%, respectively.