Algal organic matter (AOM) is considered to be threatening for the consumption of disinfectants and the formation of disinfection by-products (DBPs) during the disinfection process. Incompatible parameters in the conventional pretreatment of algal-laden water will lead to counterproductive results, such as AOM release. Therefore, the generation of AOM and its conversion to DBPs during pretreatment should be observed. The characteristics of DBPs from extracellular organic matter (EOM) and intracellular organic matter (IOM) were epitomized and simulation experiments were conducted in deionized (DI) water and source water under pretreatment conditions. Differences in DBP formation between the different backgrounds during chlorination and powdered activated carbon (PAC) treatment were investigated. Instead of monotonous excitation-emission matrix (EEM) spectra, molecular weight (MW) fractionation was simultaneously applied to elucidate the mechanisms of chlorination and PAC adsorption on AOM-based DBPs. The fluorescence regional integration (FRI) EEM results showed a clear correlation between the fluorescent properties and MW distribution of AOM. A decreasing trend was observed after a rapid increase in fluorescence intensity during the chlorination and PAC treatment of water samples in the simulation experiments in deionized (DI) water and source water. The DBP formation potential (FP) in the source water was consistent with the change in AOM during chlorination and PAC adsorption. In addition, EEM showed decent predictability of AOM-based trihalomethanes (THM) FPs (R2 = 0.77–0.99) invoking a combination with MW fractionation. Macromolecular protein compounds were highly correlated with the formation of dichloroacetonitrile (DCAN) (R2 = 0.89–0.98). These post-mortems results imply that EEM spectra are a useful tool for identifying AOM-based precursors to reveal the accurate environmental fate and risk assessments of AOM.