Rationale High‐resolution mass spectrometry (HRMS) combined with electrospray ionization (ESI) has been the most useful technique for molecular characterization of dissolved organic matter (DOM) derived from diverse sources. However, the comprehensive detection of DOM composition was hindered by ionization suppression observed in ESI sources. HRMS coupled with liquid chromatography (LC) is a potential tool for comprehensive molecular characterization of DOM. Methods The Suwannee River fulvic acids (SRFAs) and two DOM samples from seawater and refinery wastewater extracted by solid phase extraction (SPE) were analyzed by LC‐Orbitrap MS coupled with ESI. The mobile phases, solvent composition, and gradient in the LC‐Orbitrap MS analysis were optimized. Results The number of detected molecular formulae of SRFAs by online LC‐Orbitrap MS was significantly increased by approximately 40% compared to direct injection. These additional detected compounds are mainly protein and lignin‐like compounds, with a low O/C ratio and high H/C ratio. For the SRFAs, the relative standard deviations (%) of reproducibility are 5.51, 2.33, 7.97, and 1.80 for average O/C, H/C, double bond equivalent, and modified aromaticity index, respectively. Conclusions This study proposed a simple and rapid method based on LC‐Orbitrap MS for an in‐depth analysis of the molecular composition of DOM, achieving a remarkable analysis time of only 5 min per sample. The rapid method provides a dependable and efficient approach for the molecular characterization of DOM, thereby advancing our comprehension and investigation of DOM across diverse ecosystems.