The controllable synthesis of specific defective carbon catalysts is crucial for two-electron oxygen reduction reaction (2e − ORR) to generate H 2 O 2 due to the great potential applications. Herein, the defective carbon catalysts (Mo-CDC-ns) were prepared by an electrochemical activation (ECA) method with Mo 2 C/C as a parent. Electrochemical cyclic voltammetry curves, X-ray photoelectron spectroscopy, inductively coupled plasma-mass spectrometry, scanning electron microscopy, and high-resolution transmission electron microscopy confirm the evolution process of a defective carbon structure from the Mo 2 C phase in which Mo species are first oxidized to Mo 6+ species and then the latter are dissolved into the solution and defective carbon is simultaneously formed. Raman and electron paramagnetic resonance spectra reveal that the defect types in Mo-CDC-ns are the edge defect and vacancy defect sites. Compared with the parent Mo 2 C/C, Mo-CDC-ns exhibit gradually increased kinetic current density and selectivity for H 2 O 2 generation with an extension of activation cycles from 10 (Mo-CDC-10) to 30 (Mo-CDC-30). Over Mo-CDC-30, a kinetic current density of 19.4 mA cm −2 and a selectivity close to 90% in 0.1 M KOH solution were achieved, as well as good stability for H 2 O 2 production in an extended test up to 12 h in an H-cell. Graphene planes and Stone Wales 5757-carbon were constructed as basic models for density functional theory calculations. It revealed that the obtained defective structure after the removal of Mo atoms contains the double vacancy at the edge of graphene (Edge-DVC) and the topological defect on the plane of 5757-carbon (5757C-D), which show more moderate reaction free energy for forming *OOH and smaller energy barrier of 2e − ORR.