Dissolved organic matter (DOM) released by straw returning for decomposition interacts with selenium (Se) in soil, which affects the speciation distribution of Se and its bioavailability. However, the relative mechanisms involved are slightly understood. This study investigated the effects of straw-derived DOM on two levels of exogenous selenite (low-Se and high-Se treatments) in two types of soil with distinct pH. Interactions between DOM and Se were revealed through three-dimensional excitation emission matrix (3D-EEM) fluorescence spectroscopy and two-dimensional correlation spectroscopy (2D-COS). Results showed that straw amendment significantly enhanced selenite bioavailability in alkaline Lou soil regardless of Se application rates (p <
0.05). However, only the high-Se treatment generated remarkable Se content in wheat grains in acidic krasnozems (p <
0.05). Selenite predominantly incorporated with phenolic and etheric C-O functional groups of DOM in soil, which mainly existed in aromatic DOM such as humic acid (HA). Consequently, HA-Se was more likely to form in krasnozems enriched with HA. 2D-COS evidenced that HA mineralization promoted Se bioavailability in krasnozems with high-Se treatment. After selenite complexed with saturated and unsaturated aliphatic carboxyl groups (CO) of DOM, it formed Hy-Se and FA-Se in Lou soil, which could be directly absorbed by wheat roots. Therefore, the composition and functional group reaction sequences of DOM in different soils manipulated selenite bioavailability in soil. These findings could provide a basis for regulating Se bioavailability during biofortification in soils.