This study presents an overview of the changes in humic acids, which are disinfection by-product precursors in a raw water canal, Bangkok, Thailand, during different seasons. Fluorescence excitation–emission matrix (EEM) spectroscopy was employed to characterize and quantify spatial and seasonal variations of dissolved organic matter (DOM) along a raw water canal of the Metropolitan Waterworks Authority (MWA) Bangkok, Thailand. A total of 276 raw water samples were collected from 23 stations during the winter, summer and rainy season. Results indicate that hydrophobic fractions made up the majority of DOM and this water source also had high specific UV absorbance (SUVA). Seasonal variation of DOM was found to be more pronounced than geographical variation along the canal. The presence of humic substances was the highest in the rainy season due to rainfall and surface runoff, while soluble microbial by-product-like substances were found only in summer. The results provide an insight into the prediction of humic acids in source water, which benefits the MWA in being aware of seasonal variation in water quality.