Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) has been widely applied to characterize the molecular composition of dissolved organic matter (DOM) in different ecosystems. Most previous studies have explored the molecular composition of DOM focused on one or a few ecosystems, which prevents us from tracing the molecular composition of DOM from different sources and further exploring its biogeochemical cycling across ecosystems. In this study, a total of 67 DOM samples, including soil, lake, river, ocean, and groundwater, were analyzed by negative-ion electrospray ionization FT-ICR MS. Results show that molecular composition of DOM varies dramatically among diverse ecosystems. Specifically, the forest soil DOM exhibited the strongest terrestrial signature of molecules, while the seawater DOM showed the most abundant of biologically recalcitrant components, for example, the carboxyl-rich alicyclic molecules were abundant in the deep-sea waters. Terrigenous organic matter is gradually degraded during its transport along the river-estuary-ocean continuum. The saline lake DOM showed similar DOM characteristics with marine DOM, and sequestrated abundant recalcitrant DOM. By comparing these DOM extracts, we found that human activities likely lead to an increase in the content of S and N-containing heteroatoms in DOM, this phenomenon was commonly found in the paddy soil, polluted river, eutrophic lake, and acid mine drainage DOM samples. Overall, this study compared molecular composition of DOM extracted from various ecosystems, providing a preliminary comparison on the DOM fingerprint and an angle of view into biogeochemical cycling across different ecosystems. We thus advocate for the development of a comprehensive molecular fingerprint database of DOM using FT-ICR MS across a wider range of ecosystems. This will enable us to better understand the generalizability of the distinct features among ecosystems.