Crop straw return is a widely used agricultural management practice. The addition of crop straw significantly alters the pool of dissolved organic matter (DOM) in agricultural soils and plays a pivotal role in the global carbon (C) cycle, which is sensitive to climate change. The DOM concentration and composition at different soil depths could regulate the turnover and further storage of organic C in terrestrial systems. However, it is still unclear how crop straw return influences the change in DOM composition in rice paddy soils. Therefore, a field experiment was conducted in which paddy soil was amended with crop straw for 10 years. Two crop straw-addition treatments [NPK with 50% crop straw (NPK+1/2S) and NPK with 100% crop straw (NPK + S)], a conventional mineral fertilization control (NPK) and a non-fertilized control were included. Topsoil (0–20 cm) and subsoil (20–40 cm) samples were collected to investigate the soil DOM concentration and compositional structure of the profile. Soil nutrients, iron (Fe) fraction, microbial biomass carbon (MBC), and concentration and optical properties (UV–Vis and fluorescence spectra) of soil DOM were determined. Here, we found that the DOM in the topsoil was more humified than that in the subsoil. The addition of crop straw further decreased the humidification degree of DOM in the subsoil. In crop straw-amended topsoil, microbial decomposition controlled the composition of DOM and induced the formation of aromatic DOM. In the straw-treated subsoil, selective adsorption by poorly crystalline Fe(oxyhydr)oxides and microbial decomposition controlled the composition of DOM. In particular, the formation of protein-like compounds could have played a significant role in the microbial degradation of DOM in the subsoil. Overall, this work conducted a case study within long-term agricultural management to understand the changes in DOM composition along the soil profile, which would be further helpful for evaluating C cycling in agricultural ecosystems.