The electrooxidation of 5‐hydroxymethylfurfural (HMF) offers a promising green route to attain high‐value chemicals from biomass. The HMF electrooxidation reaction (HMFOR) is a complicated process involving the combined adsorption and coupling of organic molecules and OH− on the electrode surface. An in‐depth understanding of these adsorption sites and reaction processes on electrocatalysts is fundamentally important. Herein, the adsorption behavior of HMF and OH−, and the role of oxygen vacancy on Co3O4 are initially unraveled. Correspondingly, instead of the competitive adsorption of OH− and HMF on the metal sites, it is observed that the OH− can fill into oxygen vacancy (Vo) prior to couple with organic molecules through lattice oxygen oxidation reaction process, which could accelerate the rate‐determining step of the dehydrogenation of 5‐hydroxymethyl‐2‐furancarboxylic acid (HMFCA) intermediates. With the modulated adsorption sites, the as‐designed Vo‐Co3O4 shows excellent activity for HMFOR with the earlier potential of 90 and 120 mV at 10 mA cm−2 in 1 m KOH and 1 m PBS solution. This work sheds insight on the catalytic mechanism of oxygen vacancy, which benefits designing a novel electrocatalysts to modulate the multi‐molecules combined adsorption behaviors. The electrooxidation of 5‐hydroxymethylfurfural (HMFOR) process is complicated, including the HMF and OH− jointly adsorbed and coupled with each other. In this work, the role of oxygen vacancies is investigated during HMFOR. It is found that the OH− tends to fill into Vo and participate in the dehydrogenation of HMF molecules, which can improve the catalytic activity of HMFOR.