Pre-ozonation can enhance the removal of algae in source water during cyanobacterial blooms; however, little is known about the influence of the co-existing allochthonous natural organic matter (NOM) on algal removal and algal organic matter (AOM) behavior during ozonation. This study aims to elucidate in the presence and absence of allochthonous NOM and the effects of varying ozone doses on Synechococcus sp. cell removal, membrane integrity, and dissolved organic matter (DOM) release and removal. The results indicate that ozone effectively disrupted algal cell membranes, reducing algal density; however, the presence of allochthonous NOM delayed cell rupture by competing for ozone due to aromatic humic-like substances. Pterin-like and protein-like fluorescent compounds were released upon cell disruption. Due to that, excess ozone led to the oxidation of the released pterin-like compounds, with characteristic fluorescence changes correlating to ozone dosage; these changes are potential to be used as an indicator to determine the optimized ozone dosage, avoiding more adverse release of intracellular AOM to form disinfection byproducts.