Dissolved organic nitrogen derived from soluble microbial products (SMPs-DON) generated during the drinking water biological treatment process poses a great threat to water supply safety due to the potential carcinogenic risk. To further study the production mechanism and characteristics of SMPs-DON in drinking water biological aerated filtration (BAF), Illumina MiSeq sequencing is applied to characterize the microbial community. In addition, an excitation–emission matrix combined with the parallel factor model (EEM-PARAFAC) and gel filtration chromatography (GFC) are used to analyze the component and molecular weight (MW) distribution of the SMPs-DON. Results showed that the production of SMPs-DON in drinking water BAF can be explained using Illumina MiSeq sequencing from the perspective of the microbial community. Also, according to the EEM-PARAFAC analysis, the fluorescence intensity scores of fulvic-like and humic-like substances were almost unchanged, whereas the scores of protein-like substances first increased and then decreased, which was consistent with the variation in the DON concentration. SMPs produced initially primarily consisted of macromolecules with MW >20 kDa, and then they were degraded and small molecular SMPs with MW <5 kDa accumulated. This study provides theoretical guidance and technical support for ensuring drinking water safety and reducing secondary pollution risks from drinking water biological treatment.