Abstract: Dissolved organic matter (DOM) affects the degradation and transformation of pollutants in groundwater, and the changes of DOM structural composition can reflect the migration and transformation process of external pollutants. Taking groundwater from a polluted site in Shandong Province as the research object, three-dimensional fluorescence spectroscopy (EEM), synchronous fluorescence spectroscopy (SFS) and fluorescence region volume integral method (FRI) were used to analyze the composition and structure of the groundwater DOM. The role of DOM spectral information in indicating the natural attenuation effect of groundwater organic pollution was investigated in combination with the natural attenuation capacity assessment methods, i.e. hydrogeochemical index analysis method and microbiological analysis method. The results showed that the content of both humus-like and protein-like substances in groundwater increased under the persistence of organic pollution in groundwater and long-term microbial action, and the proportion of protein-like substances gradually increased with the increasingly strong biodegradation. Based on FRI region theory, a substitute characterization index was proposed to quickly assess the effect of the biodegradation in the natural attenuation of organic pollution in groundwater: the ratio of relative contents of specific fluorescence regions P_{(Ⅰ +Ⅱ)} /P_Ⅴ, that was, the ratio of the relative content of protein-like substances to humus-like substances. The larger the value of P_(Ⅰ+Ⅱ) /P_Ⅴ was, the stronger the biodegradation in natural attenuation was.