Dissolved organic matter (DOM) response to biogeochemical processes in terrestrial and aquatic ecosystems, serves as a critical component for global and regional carbon cycling. However, spectral characteristics of aqueous DOM in region-specific sites, particularly in widely distributed karst waters are not well known. Here, we examined Hongfeng Lake, Baihua Lake, and Laoma River in Guizhou Province, China, to explore the composition and source of DOM in karst lake-river systems. We analyzed UV-vis parameters (SUVA₂₅₄, SUVA₂₈₀, S_275-295, S_350-400, S_R, and E2/E3) and three-dimensional fluorescence combined with parallel factor (PARAFAC), as well as fluorescence parameters (HIX, FI, BIX, and β：α) for DOM characterization. Spearman's correlation analysis and principal component analysis (PCA) methods were employed for revealing inherent relationships among DOM parameters and primary variable contributions. The results showed that SUVA₂₅₄ and SUVA₂₈₀ values were low while E2/E3, S_275-295, and S_350-400 values were relatively high in the Hongfeng Lake, Baihua Lake, and Laomahe, indicating relatively limited proportion of aromatic components and low-molecular-size DOM. We found primary DOM components were: C1: microbially-derived humus, C2: visible-light humus and C3: tryptophan in the Hongfeng Lake and Baihua Lake, and yet C1: tryptophan, C2: fulvic acid, and C3: terrestrial humus in the Laoma River. The BIX ranged between 0.77 and 1.12, indicating that both biological and terrestrial inputs contributed to aquatic DOM. The FI values were lower than 1.4 in the lake Hongfeng and Baihua, while they varied from 1.4 to 1.9 in the Laoma River. This suggested the higher contributions of endogenous DOM in the river than that in the lakes. Aquatic β：α ranged between 0.73 and 1.10, indicating a significant contribution from newly produced DOM. The low HIX implied the limited humification in the studied karst waters. HIX was positively correlated with S_R and S_275-295 (P ＜ 0.05) and negatively correlated with FI (P ＜ 0.05), indicating that humification was constrained by DOM molecular size and sources. The homogeneity of aromatic DOM led to mutual couplings between SUVA₂₅₄ and SUVA₂₈₀. We highlighted that recently produced DOM was often biodegradable, which supported the strong correlations between BIX and β：α. The karst river exhibited more significant characteristics regarding endogenous contributions in comparison to the lakes, which might be attributing to rapid biogeochemical cycling within turbulent systems. Our results clarify the significance of terrigenous and biogenic signals in karst waters, upscaling the role of autochthonous and allochthonous carbon inputs for DOM dynamics. The study uncovers spectral characteristics of DOM composition and source in a karst lake-river system, which is expected to supplement useful datasets for global carbon cycling.