Microbially derived extracellular polymeric substances (EPSs) occupy a large portion of dissolved organic matter (DOM) in surface waters, but the understanding of the photochemical behaviors of EPS is still very limited. In this study, the photochemical characteristics of EPS from different microbial sources (Shewanella oneidensis, Escherichia coli, and sewage sludge flocs) were investigated in terms of the production of reactive species (RS), such as triplet intermediates (3EPS*), hydroxyl radicals (•OH), and singlet oxygen (1O2). The steady-state concentrations of •OH, 3EPS*, and 1O2 varied in the ranges of 2.55–8.73 × 10–17, 3.01–4.56 × 10–15, and 2.08–2.66 × 10–13 M, respectively, which were within the range reported for DOM from other sources. The steady-state concentrations of RS varied among different EPS isolates due to the diversity of their composition. A strong photochemical degradation of the protein-like components in EPS isolates was identified by excitation emission matrix fluorescence with parallel factor analysis, but relatively, humic-like components remained stable. Fourier-transform ion cyclotron resonance mass spectrometry further revealed that the aliphatic portion of EPS was resistant to irradiation, while other portions with lower H/C ratios and higher O/C ratios were more susceptible to photolysis, leading to the phototransformation of EPS to higher saturation and lower aromaticity. With the phototransformation of EPS, the RS derived from EPS could effectively promote the degradation of antibiotic tetracycline. The findings of this study provide new insights into the photoinduced self-evolution of EPS and the interrelated photochemical fate of contaminants in the aquatic environment.