An novel analytical method consisting of flow-injection sampling and chemiluminescence (CL) detection for determination of anticancer drug hydroxycamptothecinum (HCPT) is described. It is based on the CL reaction of potassium permanganate-formaldehyde-HCPT system. The formaldehyde-sensitized CL emission mechanism was proposed for HCPT-potassium permanganate (KMnO 4 )-HCOH systems by comparing the fluorescence emission with CL spectra. Illuminant was the single-state bimolecular oxygen, 1 O 2 1 O 2 ( 1 D g 1 D g ), which was from 1 O 2 ( 1 D g ) produced in the reaction system, and emits CL spectra at 639 nm. The presence of formaldehyde can sensitized CL emission, because it may be to accelerate the generation of 1 O 2 ( 1 D g ). The optimum conditions for CL emission were investigated and optimized. The CL intensity was correlated linearly with concentration of HCPT in the range of 0.010-60.0 mg L ร1 . The determination limit is 0.006 mg L ร1 . The relative standard deviation is 1.6% for 11 parallel measurements of 0.20 mg L ร1 HCPT standard solution. The proposed method has been applied for the determination of HCPT in pharmaceutical preparations, serum, and urine. The results of the quantitative analysis for HCPT studied using the developed method showed a good agreement with provided by using an official method.