Single and a few atomic-layer molybdenum disulfide (MoS2) is a promising material in the fields of hydrogen generation, battery, supercapacitor, and environmental protection, owing to the outstanding electronic, optical, and catalytic properties. Although many approaches have been developed for exfoliation of MoS2 sheets, it is still essential to develop simple, convenient, and environmental friendly exfoliation methods. More importantly, the microscopic exfoliation process and the mechanism are still not clear, limiting a deeper understanding of the exfoliation. Herein, we develop a convenient and clean method for exfoliation of the 2H phase MoS2 (2H-MoS2) deposited on an indium tin oxide (ITO) surface. Importantly, the exfoliation process is observed directly and continuously under an optical microscope to reveal the detailed exfoliation process and mechanism. As illustrated, the light illumination triggers the exfoliation of the 2H-MoS2 sheets, and the presence of water is essential in this exfoliation process. The light intensity and wavelength, humidity, and bias all affect the exfoliation process obviously. The exfoliation is caused by the vaporization of the water molecules intercalated in 2H-MoS2 interlayers. By using this method, 2H-MoS2 nanosheets with different thicknesses are prepared on the ITO substrate, and microscopic catalysis mapping of the exfoliated sheets is demonstrated with single-molecule fluorescence microscopy, revealing that the prepared thin-layer 2H-MoS2 nanosheets show improved electrocatalysis activity (roughly 20 times). Our work will not only help deepen the understanding of exfoliation process of two-dimensional nanosheets but also provide an effective tool for the in situ study of various properties of the exfoliated sheets.