The surface potential (SP) variations in mono and multilayer molybdenum disulfide (MoS2) are visualized in situ and detected using Kelvin probe force microscopy (KPFM) in different humidity conditions for the first time. N-type doping, which originates from the SiO2 substrate, is discovered in the exfoliated MoS2 and is accompanied by a screening length of five layers. The influence of water, which serves as an environmental gating for MoS2, is investigated by controlling the relative humidities (RHs) in the environmental chamber. A monotonic decrease in the SP is observed when the threshold concentration is achieved. This corresponds to the Fermi level variation, which is dominated by different processes. The results also indicate that water adsorption could result in MoS2 p-type doping and provide compensation that partially counteracts the substrate effect. Under this condition, the interlayer screening effect is influenced because of the water dipole-induced electric field. Density functional theory calculations are performed to determine the band structure variations and the interactions between water molecules and between water molecules and the MoS2 surface in mono and trilayer MoS2 under different RHs. The calculations are in excellent agreement with the experimental results. We propose that in situ measurements of the SP using KPFM under different environmental regimes is a noninvasive and effective method to provide real-time visualization and detection of electronic property variations in two-dimensional materials.