Abstract: To explore the simultaneous determination of naringenin and eriodictyol in etiolation extract of fresh- cut Chinese water- chestnut,HPLC was established by optimizing the chromatographic conditions including mobile phase,column temperature,flow rate,etc. in using Agilent Eclipse XDB- C18 column. The optimal simultaneous determination conditions were the mobile phase composed of 0.5%( v / v) acetic acid in water( eluent A) and acetonitrile( eluent B) in a linear gradient program of 0~10 min,10% ~20% B,10~15 min of 20% ~30% B,15~25 min of 30% ~40% B and 25~35 min of 40% ~50% B at a continual flow rate of 1.0 m L·min^-1at a column temperature of40 ℃,the injection volume was 10 μL,and the UV detection was performed at 280 nm,which could make naringenin and eriodictyol separate successfully. Naringenin and eriodictyol revealed high correlation between 5.60 ~28.00 μg·m L^-1( r = 1.0000) and between 3.20~16.00 μg·m L^-1( r = 0.9999) and exhibited recovery range of 99.49% ~100.71% and 98.68% ~ 101.65%( n = 6),respectively. The chromatographic conditions were simple,accurate and reliable,with good reproducibility,which can eliminate the chromatography of other substances contained and interference of other substances produced,so it can be used as a method that simultaneously determine naringein and eriodictyol in etiolation tissues of fresh- cut Chinese water- chestnut.