Human activities have greatly altered terrestrial carbon (C) dynamics associated with vegetation cover and land use changes, thereby influencing the C sink in downstream ecosystems. However, the transport and preservation of organic C from soils that experience serious erosion in the karst area are scarce, particularly at catchment scales. In this study, chemical characteristics of organic matter (OM) isolated from the topsoil, overlying water, and lake sediments, as well as subsequent source identification, were inferred from the molecular, spectroscopic, and carbon isotopic (delta C-13) signatures in a typical karst catchment, Southwestern China. The results indicated that the elemental compositions of the calcareous soil and paddy soil significantly differed from the yellow soil. High similarities existed in the fluorescence spectra of humic substances (HS) extracted from the front two soil types with those of lake sediments, indicating the homogeneous nature of OM molecular structure. The C/N ratios of six dissolved OM fractions and sedimentary HS along with delta C-13 values consistently reflected the primary terrestrial source. It was estimated to account for 60% of total organic C in sedimentary OM by end-member mixing modeling in accordance with soil erosion intensity and large recharge coefficient of this catchment. The evolution of soil loss and lake productivity can be well deduced from sediment records of organic C content, C/N ratio, and the specific information of HS. This research highlighted that the composition, source, and fate of OM in the karst lake was mainly dominated by the terrestrial C flux, rather than in-lake production. Furthermore, soil type and erosion intensity have significant effects on the nature of eroded OM and ultimate preservation.