A highly-sensitive two-dimensional high-performance liquid chromatographic (2D-HPLC) system with narrowbore reversed-phase and microbore enantioselective columns has been developed for the determination of alanine (Ala) and serine (Ser) enantiomers. The amounts of D-amino acids in biological samples are extremely low in most cases and improvement in the sensitivity is still required. For a highly-sensitive analysis, a narrowbore reversed-phase column, KSAARP (1.5 mm x 500 mm, having a wider ID than the microbore column) was used in the first dimension, and a microbore enantioselective column, KSAACSP-001S (1.0 mm x 250 mm, having a narrower ID than the frequently reported enantioselective columns) was used in the second dimension. The amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and detected by the fluorescence detectors. The 2D-HPLC system was validated using a mouse cerebrum in addition to the standard amino acids, and satisfactory calibration lines and precision results were obtained. In the mouse cerebrum, a large amount of D-Ser and a small amount of D-Ala were clearly observed. These results indicated that the present method is applicable to the determination of the trace amounts of D-amino acids in tissue samples.