The electrocatalytic oxidation reaction plays a key role in energy conversion and storage systems. In order to achieve the best energy efficiency and cost competitiveness in these systems, a comprehensive understanding of the strategic design of electrocatalysts and the underlying mechanisms is essential. Defect engineering, especially the incorporation of oxygen vacancies (OVs), has proven to be an effective electrocatalyst modification strategy. OVs can regulate the electronic structures of metal oxides and hydroxides, generate unsaturated coordination sites on the surfaces of catalysts, and act as active sites to significantly accelerate the rates of electrocatalytic reactions. In recent years, studies have shown that OVs play an important role in electrocatalytic oxidation reactions such as the oxidation of hydrocarbons, alcohols and amines. This review discusses the strategies for generating OV sites, advanced characterization techniques for identifying and analyzing OVs, and theoretical calculations to elucidate the underlying mechanisms. In addition, the application of OVs in the electrocatalytic process is particularly emphasized, which is crucial for elucidating the dynamic evolution of OVs in the reaction process and further promoting the design of efficient electrocatalytic systems. We believe that this paper will provide new ideas and ways to promote the development of new fields such as OV energy conversion and environmental protection.