Abstract: Due to the low oxidation potential and high electrochemiluminescence (ECL) quantum yield, Luminol has been widely applied in analytical and detection fields, especially in immunoassay, food safety and environmental monitoring. However, there is a lack of sufficient experimental evidence on the ECL mechanism of Luminol, which affects the further improvement of its ECL efficiency. Herein, an electrochemistry-micro tube-mass spectrometry (EC-MT-MS) device was constructed for on-line monitoring of Luminol ECL reaction. In this EC-MT-MS device, a high borosilicate glass tube with a nano tip was used as electrochemical cell and electrospray ionization source, a platinum working electrode and a platinum counter electrode were inserted into the tube and connected to a customized power station, which could simultaneously input a low voltage (volt level) for the ECL reaction and a high voltage (kilovolt level) for the formation of electrospray. Prior to the EC-MT-MS experiment, the composition of the reaction solution was optimized to obtain the highest MS signal response, and Luminol was dissolved in acetonitrile-water (4:1, V/V) solvent with ammonium acetate as the electrolyte. The concentrations of Luminol and ammonium acetate in the prepared solution were both 0.1 mmol/L, and the pH was adjusted to 11 using ammonium aqueous. The oxidation potential of Luminol was subsequently studied using a traditional three-electrode system by cyclic voltammetry in the same solution, and the determined oxidation potential for ECL reaction was 0.87 V. During the EC-MT-MS measurements, (2.5 kV+0.87 V) and 2.5 kV were input to the working and counter through the power station, respectively. A series of short-lived intermediates and products such as L₁ (m/z 198) and the luminescent precursor AP^- (m/z 180) in the Luminol ECL reaction process were successfully captured and analyzed by MS. The molecular structures of these products were characterized by tandem mass spectrometry (MSⁿ) technique. There are mainly two fragments of m/z 163, 136 present in the MS² spectrum of luminescent precursor AP^-, attributed to the (NH₂+H) and COO losses, respectively. In addition, the promotion mechanism of hydrogen peroxide (H₂O₂) for the ECL performance of Luminol was also investigated. A key reaction intermediate LO₂H^- was found in the MS spectrum at m/z 208. Meanwhile, the peak intensity of luminescent precursor AP^- significantly increased from 1.3% to 15.7%, proving that the presence of H₂O₂ effectively promoted the generation of key luminescent intermediate. Finally, by adding 1,4-benzoquinone to the Luminol-H₂O₂ system, the central role of the superoxide anion radical (O₂^·-) derived from H₂O₂ in the Luminol ECL process was confirmed. The relevant results provide a direct data reference for the study of the Luminol ECL mechanism.