The Cerrado (savanna) and Pantanal (wetland) biomes of Central Western Brazil have experienced significant development activity in recent decades, including extensive land cover conversion from natural ecosystems to agriculture and urban expansion. The Cuiabá River transects the Cerrado biome prior to inundating large areas of the Pantanal, creating one of the largest biodiversity hot spots in the world. We measured dissolved organic carbon (DOC) and the optical absorbance and fluorescence properties of dissolved organic matter (DOM) from 40 sampling locations spanning Cerrado and Pantanal biomes during wet and dry seasons. In the upper, more agricultural region of the basin, DOC concentrations were highest in the rainy season with more aromatic and humified DOM. In contrast, DOC concentrations and DOM optical properties were more uniform for the more urbanized middle region of the basin between wet and dry seasons, as well as across sample locations. In the lower region of the basin, wet season connectivity between the river and the Pantanal floodplain led to high DOC concentrations, a fourfold increase in humification index (HIX) (an indicator of DOM humification), and a 50% reduction in the spectral slope (SR). Basin‐wide, wet season values for SR, HIX, and FI (fluorescence index) indicated an increasing representation of terrestrially derived DOM that was more humified. Parallel factor analysis identified two terrestrially derived components (C1 and C2) representing 77% of total fluorescing DOM (fDOM). A third, protein‐like fDOM component increased markedly during the wet season within the more urban‐impacted region.