Oxygen vacancies play a vital role in the catalytic activity of layered double hydroxide (LDH) catalysts in wastewater treatment. However, the mechanism of oxygen vacancy-mediated LDH-activated oxygen to produce reactive oxygen species (ROS) still lacks a reasonable explanation. In this work, a tartrate-modified CuCoFe-LDH (CuCoFe/Tar-LDH) with abundant oxygen vacancies was designed, which can efficiently degrade nitrobenzene (NB) under room conditions. The technical energy consumption is 0.011 kW h L. According to the characterization and calculation results, it is proposed that oxygen vacancies are formed because of the oxygen deficiency which is caused by the reduction of the energy between the metal ion and oxygen, and the metal ion transitions to a lower state. Compared with CuCoFe-LDH, the oxygen vacancy formation energy of CuCoFe/Tar-LDH decreased from 1.98 to 1.13 eV. The O bond length adsorbed on the oxygen vacancy is 1.27 Å, close to the theoretical length of superoxide radicals (O) (1.26 Å). Radical trapping experiments and electron spin-resonance spectroscopy spectrum prove that O is an important precursor of OH. This work is dedicated to the in-depth exploration of the oxygen vacancy-mediated CuCoFe/Tar-LDH catalyst activation mechanism for molecular oxygen and the conversion relationship between ROS.