The Li–O2 batteries (LOBs) have arouse worldwide interests due to their superior energy density and environmental sustainability. The researches on Li–O2 battery electrocatalysts can contribute to the development of Li–O2 batteries. Among various catalysts, Co3O4 has arouse some interests owing to its high abundance, low cost and bifunctional activity. In this study, a vacancy engineering method was adopted to tune the contents of oxygen vacancy and metal ion vacancy in 2D micro-assembly Co3O4 nanosheets through changing annealing temperature. The synergistic effects of mesoporous morphology, oxygen vacancy and higher Co3+ content induce the best cycle performances of the Li–O2 cell, because structural defects can create a pathway for O2 to enter the interior of the 2D micro-assembly Co3O4 nanosheets, serve as active sites and thus accelerate ORR, and the diffusion of oxygen and electrolyte can be assisted by mesopores in Co3O4 nanosheets to prevent channel clogging by discharge products and facilitate rapid mass of oxygen and lithium ions, and the large surface area of nanosheets can accommodate substantial discharge products. This work offers new horizons in the consideration and design of transition-metal oxides as cathode materials for LOBs.