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Periphyton is a ubiquitous niche in aquatic environments and can be a significant source of dissolved organic matter (DOM) production and leaching, especially in such environment as the Everglades, a slow-water flow wetland in Florida, USA. We employed an array of methods, including compositional analysis, 3-dimensional excitation emission matrix (3-D EEM) fluorescence spectroscopy, and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, to perform quantitative and qualitative analyses on the DOM produced by periphyton and DOM in surrounding surface water and periphyton overlying water for comparison purposes. Higher dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) contents in periphyton pore water than surface water and periphyton overlying water indicated the remarkable contribution from periphyton-produced DOM. Higher total protein, carbohydrate, and thiol contents in periphyton pore water than in surface water and periphyton overlying water underscored the possibility of periphyton pore water DOM leached from periphyton. These results agreed with 3-D EEM and ATR-FTIR analyses that showed the prevalence of possible microbial source of periphyton pore water DOM as indicated by higher fluorescence index (FI) than surface water and periphyton overlying water. Similarly, the size-fractionated DOM from surface water demonstrated terrestrial sources, and periphyton pore water demonstrated microbial sources regardless of their differences in size based on their FI values. The types of periphyton affect the production and composition of DOM, as evidenced by higher total protein, carbohydrate, and chlorophyll-a (Chl-a) contents in floating mat on the water surface than in epiphyton attached to submerged phytoplankton, probably because the former is photo-synthetically more productive than the latter due to different light availability. This study provided fundamental information on periphyton DOM that is essential for further investigating its role in carbon cycle and its biogeochemistry.