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Straw return is an effective management practice. It not only utilizes agricultural waste but also introduces dissolved organic matter (DOM) into the soil. Selenium (Se) is an essential trace element in the human diet and contributes to the popularity of Se-enriched agricultural products in the Chinese market. Moreover, there are still some Se-enriched agricultural products that have yet to be utilized. This study investigated whether Se addition in soil caused component changes in the DOM extracted from rape straw. DOM extracted from rape straw grown in soil with four Se levels (0, 0.1, 0.5 and 1.0 mg Se kg−1 soil) was characterized by UV-Visible spectroscopy, fluorescence spectroscopy and FTIR spectroscopy. The UV-visible spectra revealed that 0.1 mg Se kg−1 soil reduced the molecular weight of DOM and caused the presence of more irreplaceable aromatic structures in the substituent groups of the DOM, while 0.5 and 1.0 mg Se kg−1 soil only reduced the DOM molecular weight. Fluorescence spectroscopy indicated that Se improved the humification degree but reduced the aromaticity of DOM. FTIR spectra proved that Se altered the contents of carboxylic acids, amino acids, alcohols and aromatic heterocycles in DOM, which were maximized in the 0.5 and 1.0 mg Se kg−1 treatment groups. We concluded that Se application could change the composition of DOM extracted from rape straw, potentially impacting the nutrient bioavailability in soil. This study provides basic data on Se-enriched rape straw utilization for eco-agriculture.