Dissolved organic matter (DOM) plays a important component in river ecosystems, and its concentration and composition changes are closely related to ecosystem functions. This study focuses on 27 Strahler 1st order headwater rivers in the Longcanggou Basin, located in the eastern edge of the Qinghai-Tibet Plateau. We utilizes DOM fluorescence characteristics to represent component features of DOM, and investigates the geographical characteristics, climate characteristics and water chemical characteristics of each river to explore the key factors controlling DOM concentration and composition in headwater rivers. The results indicated that the concentration of dissolved organic carbon (DOC) ranges from 0.35 to 1.50 mg/L, with an average value of 0.85 mg/L. In addition, the average values of fluorescence index (FI) demonstrated an average of 0.91 for agriculture group and 1.11 for forest group, while the average ratio of tryptophan to tyrosine (Trypto/Tyro) is 0.76, and the average value of the freshness index β/α is 0.61. These specific measurements and indices collectively indicate a state of limited bioavailability for proteins and a correspondingly low level of microbial activity within the studied river ecosystems.. The results showed that as altitude decreases, the DOC concentration of rivers decreases, along with a decrease in allochthonous components of DOM, while microbial activity increases (with statistical significance at P < 0.05). The results further revealed significant positive correlations between FI,β/α, as well as Ca²⁺/Mg²⁺ and the concentrations of NO₃^- (P < 0.05). The components of DOM exhibit a stability to alterations in topography, watershed area, and various climatic factors, with statistical analysis demonstrating a lack of significant influence(P > 0.05). Stepwise regression analysis reveals that elevation, slope, temperature, pH, oxidation reduction potential, DOC concentration, Ca²⁺/Mg²⁺ and NO₃^- concentration all have a significant effect on DOM components (P < 0.05). Furthermore, structural equation modeling results indicated that Ca²⁺/Mg²⁺ and the concentration of NO₃^- are the primary environmental factor driving the altitude-related variation of DOM quality in the Strahler 1st order headwater rivers. Based on the above analysis, as the altitude decreases, intensified rock weathering and increased human activities lead to an increase in NO₃^- concentration, altering the water chemistry and improving the quality of DOM components. By elucidating the spatial variation and key environmental factors influencing river DOM, this study significantly contributes to our understanding of the organic matter metabolism processes in headwater rivers. It provides valuable insights into the ecological dynamics and environmental factors shaping DOM characteristics, thereby aiding in effective management and conservation strategies for river ecosystems.