Utilization-associated products (UAPs) and biomass-associated products (BAPs) were quantified separately in this study to characterize soluble microbial products (SMPs) in a drinking water lab-scale biological aerated filter (BAF), and their basic characteristics were explored using gel filtration chromatography and three-dimensional excitation–emission matrix (3D-EEM) spectrophotometry with fluorescence regional integration analysis and parallel factor model. UAPs were observed increased with the increase of filter media depth and accumulated after BAF treatment, whereas BAPs were basically constant. 3D-EEM spectroscopy analysis result showed that tryptophan and protein-like compounds were the main components of UAPs and BAPs, and fulvic-acid-like substance was a major component of BAPs, rather than UAPs. In terms of molecular weight (MW) distribution, UAP MW presented a bimodal distribution in the range of 1–5 and >10 kDa, while BAP MW exhibited unimodal distribution with MW >20 kDa fraction accounting for more than 90 %. The macromolecules of UAPs accumulated after BAF treatment. This study provides theoretical support for in-depth study of SMP characteristics.