Estuarine systems receive large amounts of organic matter that enhance the production of greenhouse gases (GHGs), such as carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O). Despite considerable research on GHGs and dissolved organic matter (DOM) distribution in estuaries, little is known about the linkage between these gases and DOM composition. Here we evaluated the relationship between three GHGs (CO2, CH4, and N2O) and DOM composition, determined through optical properties, in Guadalete estuary (Bay of Cadiz, Spain). The partial pressure of CO2, and CH4 and N2O concentrations ranged between 332.8 and 6807.1 μatm, 19.9-6440.1 nM, and 6.8-283.9 nM, respectively. Thus, the Guadalete estuary was a source of CO2, CH4 and N2O to the atmosphere. We validated three PARAFAC components related to humic-like fluorescence from terrestrial, microbial and effluent sources, and one with protein-like material. Humic-like components accounted for 86% ± 6% of the total FDOM pool, indicating a predominantly allochthonous DOM origin. The three GHGs were significantly linked to DOC concentration and DOM composition, exhibiting different patterns in these linkages. Terrestrial and microbial humic-like substances with increasing aromaticity might enhance pCO2 in Guadalete estuary. Dissolved CH4 concentrations showed the strongest relationship with DOM composition, indicating that humic and protein-like material are linked with their distribution. In contrast, dissolved N2O was only related with the protein-like fraction and with humic-like material derived from anthropogenic activities (sewage and agriculture). Our results further indicate that a possible coupling between benthic fluxes of GHGs and DOM might be occurring in this shallow estuary. We conclude that it is important to account for DOM composition when studying GHGs distribution in estuarine systems to understand their roles and potential responses associated with climate change.