Estuaries play a pivotal role in channeling terrestrial DOM (tDOM) into the ocean, however, synchronized quantitative and qualitative characterization for the distribution and transport of tDOM across estuary is barely documented. We carried out two sampling campaigns following salinity gradient under variable fluvial discharge at a subtropical estuary. The concentration of bulk DOC and lignin was quantified, fourier transform ion cyclon resonance mass spectrometry (FT-ICR-MS) was applied to analyze the molecular composition of solid-phase-extraction DOM (SPE-DOM). The significant correlation between salinity and the concentration of DOC and lignin indicated that mixing drove the distribution of estuarine DOM. However, the principle coordinate analysis (PCoA) based on the molecular composition revealed that in situ production and microbial activities may be the main factor to regulate DOM composition within the estuary. High discharge could simultaneously increase the input from upper river to estuary and the output of estuary toward the coastal ocean with respect to the content of DOC and lignin. Lignin was removed during seaward dispersion, where microbial- and photo-degradation as well as particle sorption may play a role. Such estuarine processes could further shape the DOM matrix into more homogeneous substances, as evidenced by the decreased diversity index. An estimation showed that ~30% lignin was lost during its journey to the coastal ocean. Our results for the first time provide quantitative and qualitative evidence for the distribution and transport of tDOM across the estuary.