A reversed phase high performance liquid chromatography method for the analysis of trans-anethole,anisaldehyde and anisic acid in biotransformation liquids was developed.The separation was performed on Kromasil-100A C18 column(250 mm×4.6 mm×5 μm) using V(acetonitrile)∶ V(water)∶ V(acetic acid)=70∶ 30∶ 0.02 as the mobile phase.The flow rate was 0.8 mL/min.The detection wave length was 260 nm.The injection volume was 5 μL,and the column temperature was room temperature.Three compounds were clearly separated in 15 minute.The method showed good linear relationship,precision and repeatability.The linearity was obtained within the range of 4.080~2.652×102 mg/L for trans-anethole(R2=0.999 9),4.580~64.12 mg/L for anisaldehyde(R2=0.999 7),and 3.780~52.92 mg/L for anisic acid(R2=0.999 3).The average recoveries of trans-anethole,anisaldehyde and anisic acid were 100.34%,101.18%,and 100.31% respectively.The relative standard deviations were 0.92%,1.75%,and 0.53% respectively.The relative standard deviations of practical sample were less than 1.1%.The method was found to be simple,rapid,sensitive and accurate for the determination of trans-anethole,anisaldehyde and anisic acid in biotransformation liquids.