In order to explore the three-dimensional fluorescence characteristics of DOM during the preoxidation process of high algae-laden water with ClO2, the three-dimensional fluorescence spectroscopy technique was used to qualitatively investigate the changes of the composition of intracellular and extracellular organic matters in algae, and the quantitative analysis of changes in DOM composition was performed by fluorescence regional integral method. The results showed that the IOM fluorescence spectrum of the raw water containing high concentration of Microcystis aeruginosa had four distinct fluorescence peaks, which were protein fluorescence peaks, humic acid fluorescence peaks, fulvic acid fluorescence peaks, and soluble microbial metabolites peaks that were represented mainly by tyrosine-like substances and tryptophan-like proteins with 14.52%, 48.27%, 16.22% and 20.99%, respectively. While the EOM fluorescence spectrum had only one significant fluorescence peak with 63.14% that was mainly behalf of soluble microbial metabolites, and algae organics were mainly contained in IOM. The removal rate of chlorophyll a reached to 73.58% within 3 minafter preoxidation process with 0.5 mg·L-1 ClO2. During this process, substances in the regions Ⅰ, Ⅱ, and Ⅲ represented by amino acids, proteins, and fulvic acid respectively were released to outside of the algal cell membrane, and then were oxidized to non-biological humus by ClO2. Owing to algal cells burst constantly, IOM was released into the water during the ClO2 preoxidation process, leading to the results that the total response value of EOM increased by 54.89% within 3 min and the total response value of IOM decreased by 51.50%. Therefore, the drinking water treatment of high algae-laden water should pay close attention to the release of IOM from the algae cells. The appropriate preoxidation time and dosage should be properly selected, and the release of IOM should be reduced as much as possible under the premise of effective algae removal.This study will provide further theoretical and scientific basis for the removal of algae by pre-oxidation in drinking water and the control of algae disinfection by-products.