Global food security and climate mitigation goals are placing unprecedented demands on agricultural systems to simultaneously improve soil productivity and reduce carbon emissions. Spent mushroom substrate (SMS), the mushroom industry’s principal waste stream, offers considerable recycling potential, yet its influence on dissolved organic carbon (DOC) chemistry and soil aggregate stability remains unclear. We tested four SMS return regimes on a medium-textured fluvo-aquic soil: CK, 0 t·ha⁻¹; ORS, 22.5 t/ha; ERS, 22.5 t/ha; and SRS, 45 t/ha in total, with 22.5 t/ha applied per SMS return event. It was found that SMS improved soil structural stability across all regimes, with SRS delivering the strongest effects. Compared with CK, SRS raised the proportions of >2 mm and 0.25–2 mm aggregates by 31.62% and 33.42%, while the mean weight diameter (MWD) and geometric mean diameter (GMD) increased by 23.25% and 22.68%. SMS also elevated aromatic carbon abundance, DOC concentration, UV254, and SUVA254. Fluorescence EEM-PARAFAC resolved DOC into three component: namely, two humic-like and one protein-like, and SMS expanded the relative contribution of the humic-like C1 fraction. Overall, under the tested fluvo-aquic soil and wheat–maize rotation conditions, SMS return was associated with changes in DOC composition, higher aggregate stability, and greater aggregate-associated carbon accumulation. These findings suggest that SMS return may be a promising strategy for improving soil structure and recycling agricultural waste under similar field conditions, but its broader applicability requires further validation.