The characteristics of dissolved organic matter (DOM) and its relationships with other environmental factors are beneficial for comprehending water pollution in watersheds. This study aimed to improve our understanding of the association of DOM with water quality by connecting the spectroscopic characteristics of DOM with land cover and land use (LCLU). Clustering the tributaries of the Miho upstream watershed according to LCLU resulted in Clusters 1 and 2 having a large proportion of farmland and a large forest area, respectively. Various fluorescence indices derived from fluorescence excitation-emission matrix spectra revealed that livestock effluent resulted in the enrichment of autochthonous organic matter of algal or microbial origin in catchment areas with a high proportion of farmland. Furthermore, to analyze water quality changes according to the land-use characteristics, the water quality and spectroscopic characteristics of DOM were utilized based on the period of farmland use. Further correlation analysis indicated a high correlation between the fluorescence index (FI) in Cluster 1 and organic matter parameters and nitrogenous pollution (Total nitrogen (TN), Dissolved total nitrogen (DTN) and Nitrate nitrogen (NO₃-N)) (planting season, r = 0.991, post-planting season, r = 0.971). This suggests that the FI can be used as a surrogate to estimate the degree of water pollution in watersheds largely affected by land uses related to agricultural activity and the livestock industries.