The aim of this study was to reveal the biotransformation of human-impacted riverine dissolved organic matter (DOM) using well-controlled bioassay tests, with a focus on the biodegradation of the structural and functional makeup. Results of three-dimensional fluorescence excitation emission matrix with parallel factor analysis (EEM-PARAFAC) showed that humic substances derived from human activities (i.e., anthropogenic humic-like substances) were of higher biodegradation potentials than terrestrial and microbial humic-like substances. In addition, the biodegradation finally led to an increase of the percentage contribution of fluorescent DOM to the total DOM. Characterization by nuclear magnetic resonance (NMR) spectroscopy indicated that large amounts of the structural components of the DOM were removed within 3 h of biodegradation. Characterization by X-ray photoelectron spectroscopy (XPS) further revealed that the riverine DOM contained various functional groups that underwent different biotransformation mechanisms. The XPS data also indicated the appearance of newly generated, oxygen-rich functional groups upon biodegradation and the disappearance of nitrogen-containing groups as a result of hydrolysis and nitrification of organic and/or ammonium nitrogen.