Oxygen vacancy (Ov) is known to act as an active center of the metal oxide. Quantification of surface Ov is vital for understanding the quantitative structure-activity relationship. Facile quantification characterization of surface Ov is highly desirable but still challenging. In this study, we presented a simple colorimetric method for rapidly quantifying surface Ov. As an example of metal oxide nanoparticles, Co3O4 was used to catalyze the 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 colorimetric reaction. It was found that the absorbance of the TMB-H2O2 system was dependent on the surface Ov amount in Co3O4. The investigation of the mechanism showed that the Ov-dependent absorbance would be attributed to the activity of surface Ov to easily adsorb and dissociate H2O2 into a hydroxyl radical (•OH). The absorbance signal of the TMB-H2O2 system acted as a probe to estimate the surface Ov. This colorimetric measurement could be completed in less than 20 min. The Ov concentrations obtained by the proposed colorimetric method matched well with those obtained by X-ray photoelectron spectroscopy. This method does not require any complex operation and expensive equipment and can be performed in any ordinary chemical laboratory. So, this colorimetric method is expected to become an alternative approach for quantifying the surface Ov in metal oxide nanoparticles. This method will provide essential insights into the rational design and application of Ov.