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[Display omitted] • Humic-like fluorescence serves as a potential surrogate of TOC regardless of land use. • Protein-like fluorescence shows a strong correlation with nitrogen pollution in urban watershed. • The impact of land use is more significant than that of hydrological conditions. • High TP levels in agricultural watersheds can be detected using protein-like fluorescence. • FI serves as a universal indicator of microbial pollution in watersheds with diverse land use. Fluorescence spectroscopy has demonstrated its potential for assessing organic matter (OM) –related water quality (WQ) and identifying anthropogenically driven pollution sources within watersheds. This study involves a comparison of spatial variations in general WQ parameters and dissolved organic matter (DOM) fluorescence characteristics between two adjacent watersheds characterized by disparate land uses (i.e., urban and agricultural). The investigation encompasses both rain and non-rain events to evaluate the diagnostic utility of DOM fluorescence in monitoring WQ fluctuations. In the urban watershed, variations were predominantly influenced by wastewater effluents, leading to noticeable changes exclusively downstream from sewage treatment facilities, regardless of distinct hydrological conditions. In contrast, the agricultural watershed exhibited more extensive spatial variations, suggesting a prevalence of non-point source pollution stemming from agricultural and livestock activities. The humic-like fluorescent components (C1 and C2) exhibited a robust positive correlation with total organic carbon (TOC), unaffected by varying hydrological conditions and catchment land use. Conversely, the assessment of nutrient levels utilizing the protein-like component (C3) was considerably influenced by catchment land use. A stronger positive correlation between C3 and total nitrogen (TN) was observed in the urban watershed compared to the agricultural watershed. Moreover, the fluorescence index (FI) demonstrated a substantial positive relationship with TN in both watersheds, indicating the dominance of microbial sources in nitrogen pollution for both watersheds. No significant differences were found in the variation trends of WQ and DOM fluorescence between non-rain and rain events. This study emphasizes that catchment land use plays a pivotal role in influencing the associations between fluorescence indices and WQ parameters. Our findings validate the robust potential for replacing conventional WQ parameters with DOM fluorescence indices in watersheds characterized by distinct land uses. [ABSTRACT FROM AUTHOR]