The reaction mechanism of non-aqueous Li-O2 batteries is based on the deposition and decomposition of Li2O2. The polarization of Li-O2 batteries can be rapidly increased by operation under a high rate condition, resulting in the early capacity fade of the cells. Therefore, a well-designed catalyst with a unique structure and excellent catalytic ability is an important way to boost the round-trip performance of Li-O2 batteries, especially under high current density. In this work, a unique nanoflower structure assembled with Co3O4 nanosheets is synthesized by using 2-methylimidazole (2-MIM) as a structural directing agent. X-ray photoelectron spectroscopy (XPS) and Raman spectra reveal abundant oxygen vacancies on the surface of the Co3O4 nanoflower, which are beneficial for oxygen reduction and evolution reactions and long round-trip lifetime. Density functional theory results demonstrate that Co3O4 catalyst with oxygen vacancies could promote the wetting of Li2O2 on substrate and formation of a Li2O2 nanofilm, thereby boosting the discharge capacity of Li-O2 batteries. On account of the synergistic effect of abundant oxygen vacancies, the unique structure, and excellent oxygen evolution reaction, Co3O4 nanoflower-based cells could deliver ultralong lifetime of 276 and 248 cycles with a discharge capacity of 1000 mAh g−1 under charge/discharge current densities of 0.5 A g−1 and 1 A g−1, respectively. This study has shed light on a new strategy for catalyst preparation for long lifetime Li-O2 batteries.