MoS₂ nanosheets (MoS₂ NSs), which exhibited peroxidase-like activity, were prepared using the hydrothermal method. The microstructure and element speciation of the MoS₂ NSs were characterized using transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS), respectively. The obtained MoS₂ NSs could catalyze the H₂O₂-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to form blue oxidized TMB (ox TMB) with an absorption peak at 652 nm. The effects of MoS₂ NS concentration, incubation time, temperature, and acidity on the color development of TMB were investigated. Under the optimal conditions (1.0 μg mL⁻¹, 40 min, 40 °C, pH 4.0), the increase in absorbance, which was induced by MoS₂ NS-catalyzed TMB oxidation, enabled the detection of H₂O₂ ranging from 2.5 μmol L⁻¹ to 300 μmol L⁻¹. As H₂O₂ is one of the products of glucose oxidase (Gox)-catalyzed glucose oxidation, a colorimetric method was further established for measuring the concentration of glucose. The absorbance response was linearly dependent on glucose concentration of 2.5–200 μmol L⁻¹, with a detection limit of 2.1 μmol L⁻¹. The proposed method was applied to the assay of glucose in urine samples with satisfactory results.