Surface oxidation engineering is an effective strategy to construct nanomaterials with enhanced biocatalytic activity. In this study, a facile one-pot oxidation strategy was proposed to synthesize partially oxidized molybdenum disulfide nanosheets (ox-MoS2 NSs), which exhibit good water solubility and can be used as an excellent peroxidase substitute. Under the oxidation process, Mo–S bonds are partially broke and S atoms are replaced by excess oxygen atoms, and the released abundant heat and gases efficiently expended the interlayer distance and weaken the van der Waals forces between adjacent layers. Porous ox-MoS2 NSs can be easily exfoliated by further sonication, and the nanosheets exhibits excellent water dispersibility and no obvious sediment appear even after store for months. Benefiting from the desirable affinity property with enzyme substrates, optimized electronic structure and prominent electron transfer efficiency, the ox-MoS2 NSs exhibit enhanced peroxidase-mimic activity. Furthermore, the ox-MoS2 NSs catalyzed 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation reaction could be inhibited by the redox reaction that take place between glutathione (GSH) as well as the direct interaction between GSH and ox-MoS2 NSs. Thus, a colorimetric sensing platform was constructed for GSH detection with good sensitivity and stability. This work provides a facile strategy for engineering structure of nanomaterials and improving enzyme-mimic performance.