In this study, attapulgite was used as an additive for exploring the effects on biogas production during anaerobic co-digestion of rice straw and cattle dung. Spectroscopic techniques were applied to investigate the structural characteristics, components and evolution of dissolved organic matter (DOM). The results indicated that substrate yielded totals with the addition of attapulgite (A1 −A4) were higher that these without the addition of attapulgite (E1 − E4), about 10.205 − 15.553 L biogas for the A1 −A4 substrates, and 6.142 − 10.163 L of biogas for the E1-E4 substrates. Biogas yields of volatile solid (VS) were 210.0, 272.7, 303.4 and 254.6 mL/g for A1-A4, and 166.5, 129.0, 216.5, and 187.4 mL/g for E1-E4, respectively. Six components could be obtained by parallel factor (PARAFAC) analysis, including three protein-like and two fulvic-like substances and one humic-like substance. The maximum fluorescence intensity (Fmax) values were notably lower in E1-E4 than in A1-A4. These fluorescent DOM components played an extremely important role in the conversion of organic matter during anaerobic digestion, especially protein-like components. The results of two-dimensional correlation spectroscopy (2D-COS) analysis indicated that the preferential sequence of change in fluorescent components and functional groups can greatly affect the biogas production and the degradation and utilization of organic matter in the presence or absence of attapulgite. Various trace elements (Fe3+, Al3+, Ca2+ and Mg2+) and large specific surface area in attapulgite promote the growth of methanogenic bacteria and accelerate the progress of methane generation, which is conducive to the conversion of DOM, thereby increasing the production of biogas and methane.