In order to improve the understanding of the global carbon cycle and the stability of karst carbon sinks, it is necessary to better understand the source, distribution and transformation characteristics of organic matter (OM) in aquatic ecosystems. Here, stable isotope ratios (δ13C and δ15N), elemental analysis (C/N ratios), and lipid biomarkers were analyzed for dissolved organic matter (DOM) (<0.7 μm), particulate organic matter (POM) (>0.7 μm) of water, and organic matter from sediment cores (SCOM) to identify the sources, distribution, and transformation of OM in a subtropical karst reservoir. The results showed that short-chain (C14–20) n-alkyl lipids were more abundant than long-chain (C21–34) n-alkyl lipids in both the DOM and SCOM samples, indicating that bacteria were the primary sources of these lipids, while terrestrial organic matter (OM) made only a minor contribution to the n-alkyl lipid pool, and aquatic plants (macrophytes) OM contributed major contribution to the n-alkyl lipid pool in POM. Microbial activity and lipid degradation were more pronounced in the DOM. Furthermore, terrigenous and macrophyte-derived lipids were found to be more abundant in POM than in DOM and SCOM, suggesting that they are relatively resistant to degradation compared with phytoplankton-derived OM.