Abstract: Adopting proper pretreatment way according to the characteristics of chestnut, a high performance liquid chromatographic method was developed for determination of glyphosate in chestnut sample, and the dependability of the method was analyzed and validated. Glyphosate was separated from chestnut by water extraction assisted with ultrasonic wave and chloroform liquid-liquid extraction, and then purified by C18 column. The derivative of the extract and 9-fluorenylmethyl chloroformate was separated by anion-exchange column, and then detected by fluorescence detector. The method is proved to be rapid, sensitive and accurate with limit of detection 0.005 μg/kg, and the mean spiked recovery rates range from 76.7% to 98.3% with RSD between 3.27% and 7.77%. It accords with China national standard (GB) on detection of pesticide residue in foods, which can be used for supervising food safety.