Display omitted •We developed a method to generate micromolar level O2∙- in aqueous solution.•O2∙- was confirmed by the UV spectra under pulse radiolysis and NBT.•We constructed an in situ spectroscopy to study the reaction of O2∙- and CCl4.•CCl4 reacts slowly with O2∙- than with ·OH/SO4∙-.•Nucleophilic substitution is the major pathway for the reaction of O2∙- and CCl4. Contemporary studies emphasize that superoxide radical (O2∙-) exhibits the potential to degrade organic contaminants, but practical application of this radical in engineered waters require an in-depth understanding of its kinetic profiles in a quantitative way. Here, we developed, for the first time, a convenient and reliable approach to generate micromolar level O2∙- in aqueous solution by photolysis of formate and H2O2. The presence of O2∙- was confirmed by comparing the UV spectra under pulse radiolysis and chromogenic reaction. We then constructed an in situ long-path spectroscopy to investigate the kinetics and mechanisms of O2∙--mediated degradation of carbon tetrachloride (CCl4), a halogenated model contaminant. The rate constant for the reaction of O2∙- and CCl4 was determined to be 478 M−1 s−1. In addition, we employed the transition state theory to model the reaction rate constants. Both results show that O2∙- exhibited low reactivity towards CCl4 with bimolecular rate constant lower by at least one order of magnitude than those radicals generated in typical advanced oxidation processes such as hydroxyl and sulfate radicals. Our results also indicate that nucleophilic substitution is the major pathway, and the solvation effect plays an important role in the reaction. The complementary experimental and theoretical approaches provide a mechanistic basis for better understanding aqueous–phase O2∙- chemistry and a holistic evaluation on the application of O2∙- for the degradation of organic contaminants of emerging concern.