The differences in the structure and component characteristics of partial nitrification biofilms between autotrophic and heterotrophic conditions were investigated in this work. Three-dimensional excitation-emission matrix fluorescence spectroscopy (EEM), fluorescence staining, and confocal laser scanning microscopy (CLSM) were used to determine differences in the architecture and extracellular polymeric substance (EPS) distribution of the autotrophic and heterotrophic biofilms. Partial nitrification was successfully achieved, and the results demonstrated that an appropriate amount of organic carbon (chemical oxygen demand (COD)/N = 2.6) is advantageous for obtaining better partial nitrification. The final ammoniation and nitrosation rates achieved were 97 and 99 %, respectively. Proteins (PN) and polysaccharides (PS) were dominant in the tightly bound EPS (TB-EPS) of autotrophic and heterotrophic biofilms, with PN/PS ratios of 0.96 and 0.69, respectively. Proteins, lipids, α-D-glucopyranose polysaccharides, and nucleic acids were mostly present within the layers of biofilms, but they were distributed in the upper-middle portion of the autotrophic biofilm and increased with depth from the upper layer in the heterotrophic biofilms.