Abstract: Dissolved organic matter (DOM) is the most reactive fraction of the soil organic matter pool; that is, its structural characteristics affect its biodegradation and flux. Acid rain has deleterious effects on terrestrial ecosystems, including soil and surface water acidification, which also changes the composition of leaching DOM. In this work, we used three-dimensional excitation/emission matrix spectroscopy and gel permeation chromatography to investigate the fluorescence characteristics and molecular weight distribution of DOM leached by simulated acid rain (SAR) from red soil. Samples were taken from red soil under 22 years of five continuous fertilization treatments: chemical fertilizer (NPK), NPK plus organic manure (NPKM), organic manure (M), without fertilizers (CK), and fallow (F). Results from the fluorescence regional integration analysis showed that humic acid–like substances (Region V) in the NPKM, M, and F treatments were greater than those in the CK and NPK treatments at SAR pH 3.5 and 2.5, but the tryptophan-like compounds (Region II) in the NPKM, M, and F treatments were less than those in the CK and NPK treatments. The results of excitation/emission matrix–fluorescence regional integration and gel permeation chromatography analysis showed that the leaching DOM from NPKM, M, and F treatments was relatively stable and had a higher molecular weight than that from NPK and CK treatments at SAR pH 3.5 and 2.5. Results suggest that the structure and composition of the leaching DOM not only depend on the acid rain pH but also on the fertilizer treatments in soil.