The source of Yangtze River and Lancang River are important water sources and natural barriers of aquatic ecosystems in the lower part of the river basin. In recent years, the ecosystems and habitats of the two source areas are facing great challenges in terms of ecosystems and habitats protection, driven by the superimposed effects of climate change and human activities. Chromophoric dissolved organic matter (CDOM), as an important component of aquatic ecosystems, plays an important role in revealing the changes and responses of aquatic ecosystems. Therefore, in this study, the spectral characteristics of CDOM in the main streams and tributaries of the Yangtze River and Lancang River during the ice-melting and water-abundance periods were systematically explored by combining ultraviolet-visible (UV-Vis) spectroscopy and 3D fluorescence spectroscopy, revealing the compositional characteristics, sources and differences of CDOM in the two source areas, aiming to provide scientific basis for revealing the carbon cycle, the migration and transformation process of organic matter in the hinterland of the Tibetan Plateau, as well as for evaluating the degree of human influence in the source areas. The results showed that 1) the absorption coefficients _α254 and DOC in the Yangtze River and Lancang River sources during the ice ablation period were higher than those in the abundant water period, and the spectral parameters E2/E3 and SUVA254 indicated that the degree of humification, aromaticity, and the relative molecular mass of CDOM in the two source regions were higher overall, and all of them showed the characteristics of a strong land source of organic matter. 2) A total of eight fluorescence fractions were extracted from the two source areas, all of which were dominated by land-source-like humic substances. Among them, the CJ-C1 (260nm/500nm) and CJ-C4 (290nm/400nm), CJ-C2 (260nm/420nm) and CJ-C3 (350nm/450nm) fractions of the Yangtze River source were UVA-like humic substances, humic substances, and UVC-like humic substances, and were mainly dominated by the CJ-C2 (36.4%) and CJ-C4 ( 24.4%) fractions; LCJ-C1 (255nm/480nm), LCJ-C2 (255nm/400nm) and LCJ-C3 (330nm/440nm) of the Lancang River source were UVA-like humic substances, humic substances and UVC-like humic substances, respectively (a total of 83.4%), and LCJ-C4 (280nm/355nm) was a humic substance like tryptophan (16.6%), indicating that the CDOM composition of the Lancang River source was also affected by human activities. 3) The mean values of fluorescence index (FI) and autochthonous index (BIX) of the Yangtze River source were 1.36 and 0.26, respectively, which were lower than those of the Lancang River source (the mean value of FI was 1.45, and the mean value of HIX was 0.52), which indicated that the terrestrial humus characteristics of the CDOM of the Yangtze River source were higher than those of the Lancang River source; the CDOM of the two source areas were mainly derived from the organic matter produced by terrestrial water and soil erosion or through atmospheric deposition and burial in glacial permafrost. The CDOM of both source areas is mainly derived from the organic matter produced by land soil erosion or buried in glacial permafrost through at`1mospheric deposition, but the CDOM of the Lancang River source is also affected by the input of organic matter from agriculture and livestock in the basin.