Abstract: In order to simultaneously detect the usage of L-phenylalanine and the production of 2-phenyl-ethanol in Hypoxylon sp. fermented liquid, an HPLC external standard method was developed. The method chromatographic conditions were as follows:Chromatographic column:C₁₈ (5 μm, 250 mm×4.6 mm), Column temperature 30℃, the injection volume 10 μL, flow rate 0.7 mL/min, DAD detector, detection wavelength 258 nm, mobile phase A 0.6% acetic acid solution, mobile phase B methanol.The results indicated that good linearities between concentration and peak area were exist for L-phenylalanine and 2-phenylethanol with correlation coefficients greater than 0.999 in the range of 1~5 and 1.02~5.1 g/L, respectively.The detection limit and the quantitative limit of L-phenylalanine were 1.023 and 3.409 mg/L, respectively. The detection limit and the quantitative limit of 2-phenylethanol were 0.567 and 1.890 mg/L, respectively. The spiked recoveries of L-phenylalanine were in the range of 99.16%~101.58% with relative standard deviations (n=6) of 0.67%. The spiked recoveries of 2-phenylethanol were in the range of 98.53%~101.06% with relative standard deviations (n=6) of 1.22%.The method was simple width high accuracy and sensitivity. It was suitable for simultaneously determining the L-phenylalanine and 2-phenylethanol in the Hypoxylon sp. fermented liquid.