A two-dimensional high-performance liquid chromatographic (2D-HPLC) system using the pre-column derivatization with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) has been developed for the determination of alanine (Ala), aspartic acid (Asp) and serine (Ser) enantiomers. For the precise determination of trace amounts of D-amino acids in complex matrices, a highly sensitive and selective method is required. In the present study, amino acids were derivatized with DBD-F for the sensitive fluorescence detection and were analyzed by a selective 2D-HPLC system combining a reversed-phase column (Singularity RP18, 1.0 x 500 mm) and an enantioselective column (Singularity CSP-001S, 1.5 x 250 mm). The established system was successfully applied to the chiral amino acid analyses of Japanese traditional amber rice vinegar, human plasma and human urine samples. In the Japanese traditional amber rice vinegar and human urine, relatively high amounts of all the target D-amino acids were observed (%D=5.8-37.7), while trace amounts of D-Ser and D-Ala were found in human plasma (%D=0.5-2.1).