Colorimetric assays have drawn increasing research interest with respect to the quantitative detection of hydrogen peroxide (HO) based on artificial enzymes because of their advantages with respect to natural enzymes, including design flexibility, low cost, and high stability. Regardless, the majority of the artificial enzymes exhibit low affinity to HO with large Michaelis-Menten constants (). This indicates that the catalytic oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to blue-colored oxTMB requires a high HO concentration, hindering the sensitivity of the colorimetric assay. To address this problem, novel reduced CoO nanoparticles (R-CoO) have been synthesized in this study via a step-by-step procedure using ZIF-67 as the precursor. R-CoO exhibits a considerably enhanced peroxidase-like activity when compared with that exhibited by pristine CoO (P-CoO). The catalytic process in the case of R-CoO occurs in accordance with the typical Michaelis-Menten equation, and the affinity of R-CoO to HO is apparently higher than that of P-CoO. Furthermore, the density functional theory calculations revealed that the introduction of oxygen vacancies to R-CoO enhances its HO adsorption ability and facilitates the decomposition of HO to produce ·OH radicals, resulting in improved peroxidase-like activity. A simple and convenient colorimetric assay has been established based on the excellent peroxidase-like activity of R-CoO for detecting HO in concentrations of 1-30 μM with a detection limit of 4.3 × 10 mol/L (S/N = 3). Furthermore, the R-CoO-based colorimetric method was successfully applied to glucose detection in human serum samples, demonstrating its potential for application in complex biological systems.