Organic chloramines posed significant risks to drinking water safety. However, the formation mechanism of algae-derived organic chloramines remained unclear. In this study, it was observed that pre-oxidation of algal suspensions increased organic chloramine formation during chlorination. Compared to KMnO4 pre-oxidation, O3 significantly increased the organic chloramine formation potential of algal suspensions. Characterization was performed with size exclusion chromatography-multiple detectors (SEC-MDs) to better understand the organic chloramine formation mechanism. The results revealed that low molecular weight proteins (AMW ≤ 0.64 kDa) were the main precursors of organic chloramines after conventional water treatment processes. We then focused on 14 essential amino acids involved in protein formation. Their concentrations and organic chloramine formation potentials were determined, based on which the theoretical organic chloramine formation potentials of the studied samples were evaluated. However, dramatic gaps between theoretical and experimental organic chloramine formations were observed, which suggested that not all organic nitrogen could react with chlorine to form organic chloramine. The condensed dual descriptor (CDD) was calculated to predict the electrophilic substitution reaction sites on peptides. Furthermore, the activation barrier of each proposed reaction was computed to confirm that the reaction sites for chlorine were located on amino groups. This study clarified the formation mechanism of algal-derived organic chloramines, which could provide a powerful theoretical foundation for controlling organic chloramine formation in drinking water processes.