To enhance the catalytic activity of two-dimensional layered materials as versatile materials, the modification of transition metal dichalcogenide nanosheets such as MoS
2
by doping with heteroatoms has drawn great interests. However, few reports are available on the study of the enzyme-like activity of doped MoS
2
. In this study, a facile in situ hydrothermal method for the preparation of various ultrathin transition metals (Fe, Cu, Co, Mn, and Ni) doped MoS
2
nanosheets has been reported. Through the density functional theory (DFT) and steady-state kinetic analysis, the Co-doped MoS
2
nanosheets exhibited the highest peroxidase-like catalytic activity among them. Furthermore, a typical colorimetric assay for H
2
O
2
was presented based on the catalytic oxidation of colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue product (oxTMB) by Co-MoS
2
. The proposed colorimetric method showed excellent tolerance under extreme conditions and a broad linear range from 0.0005 to 25 mM for H
2
O
2
determination. Concerning the practical application, in situ detection of H
2
O
2
generated from SiHa cells was also fulfilled, fully confirming the great practicability of the proposed method in biosensing fields.
Supplementary Information
The online version contains supplementary material available at 10.1007/s10853-022-07201-z.