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A new method was developed to separate epinephrine enantiomers derivatized with fluorescent chiral reagent, R-(-)/S-(+)-4-(N, N-dimethylaminosulfonyl)-7-(3-iso-thiocyanatopyrrolidino)-2, 1, 3-benzoxadiazole (DBD-PyNCS), by reversed-phase high performance liquid chromatography, on a Diamonsil C18 column(150 mm x 4.6 mm i.d., 5 microns), with a mobile phase of water-acetonitrile(72:28, volume ratio) and a fluorescent detector at the excitation wavelength of 460 nm and the emission wavelength of 550 nm. The column was eluted at 40 degrees C, with a flow rate of 1 mL/min. At first, let the fluorescent chiral reagent(36 mmol/L, 10 microL) react with epinephrine enantiomers(1 mmol/L, 10 microL), and then 10 microL 20% pyridine acetonitrile solution was added. The resulting mixture was stirred for 1 min and kept at 65 degrees C for 35 min and then a 10 microL aqueous of the diastereomeric derivatives was injected directly into the chromatograph. The diastereomeric compounds were efficiently separated. The Rs value was 2.6 with alpha of 1.07. The k's of L-epinephrine and D-epinephrine were 17.11 and 18.13 respectively. The retention times of L-epinephrine and D-epinephrine were 30.24 min and 31.94 min respectively. The linear range was 0.07 g/L -0.50 g/L (r = 0.9995). The RSD was 4.3%(n = 7). The detection limit was 12 g/L(S/N = 3).