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Abstract: Dissolved organic matter (DOM) in refinery wastewater is an extremely complex mixture of various organic compounds. Using mass spectrometry, it is impossible to characterize all of the DOM molecules with only one ionization source. In this study, negative-ion, electrospray ionization (ESI), positive-ion ESI, and positive-ion atmospheric pressure photoionization (APPI) were coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to analyze the molecular composition of DOM in a refinery wastewater stream during the treatment process. There were obvious differences in the heteroatom composition, number of DOM constituents, and chemical properties in refinery wastewater under the three ionization modes. Acidic CHO and CHOS compounds detected by (−)ESI, basic CHN and CHON compounds detected by (+)ESI, and hydrocarbons detected by (+)APPI were analyzed to determine the molecular transformations that occurred during treatment. In an anaerobic biological treatment process, acidic CHO and CHOS compounds with a high oxygen content were preferentially removed, and acidic CHO and CHOS compounds with a low oxygen content were produced. In an aerobic biological process, acidic CHO and CHOS compounds with a low oxygen content were preferentially removed, and acidic CHO and CHOS compounds with a high oxygen content were produced. The whole biological treatment process has a poor removal efficiency for CHN and CHON compounds, and hydrocarbons. An activated carbon (AC) adsorption process removed different heteroatom compounds mainly with a low oxygen content for acidic and basic compounds. The transformation mechanism of CHO and CHOS compounds in the biological treatment process was analyzed by the Kendrick mass defect (KMD) theory and a mass difference network analysis. In the anaerobic process, large amounts of oxygenated CHO and CHOS compounds were degraded by decarboxylation, deoxydation, demethoxylation, and dehydration reactions, and converted to lower oxygen content compounds. In the aerobic processes, these low oxygen CHO and CHOS compounds mainly underwent carboxylation and oxidation reactions. This study determined the transformation characteristics and mechanisms of different types of organic compounds in refinery wastewater during the treatment process. The results provide guidance for the design and optimization of technologies for refinery wastewater treatment.