Camptotheca acuminata Decne is an important medicinal plant that contains various cytotoxic alkaloids, such as camptothecine (CPT) and 10-hydroxycamptothecine (HCPT). A rapid and sensitive liquid chromatography with fluorescence detection (LC-FLD) method for the quantification of CPT and HCPT is described. The separation was carried out on a DL-Cl8 column (4.6×150mm, 5μm), with the mobile phase of acetonitrile-sodium dihydrogen phosphate buffer (10mm) using an gradient elution at the flow rate of 0.6mL/min. The LC-FLD method was validated for linearity, sensitivity, accuracy and precision, and then used to determine the content of the above components. The lower detection limits of CPT and HCPT were 0.4 and 0.1ng/mL, respectively. The precision was <1.58% and the mean recovery of the analytes was 96.0-98.6%. The LC-FLD method was successfully applied to determine CPT and HCPT in real samples including C. acuminate, HCPT injection and rat plasma.