Understanding the process of charge generation, transfer, and diffusion between two-dimensional (2D) materials and their supporting substrates is very important for potential applications of 2D materials. Compared with the systematic studies of triboelectric charging in a bulk sample, a fundamental understanding of the triboelectrification of the 2D material/insulator system is rather limited. Here, the charge transfer and diffusion of both the SiO surface and MoS/SiO interface through contact electrification and frictional electrification are investigated systematically in situ by scanning Kelvin probe microscopy and dual-harmonic electrostatic force microscopy. Different from the simple static charge transfer between SiO and the PtSi alloy atomic force microscope (AFM) tip, the charge transfer between the tip and the MoS/SiO system is complicated. Triboelectric charges, generated by contact or frictional electrification with the AFM tip, are trapped at the MoS/SiO interface and act as floating gates. The local charge discharge processes can be obtained by monitoring the surface potential. The charge decay time (τ) of the MoS/SiO interface is one (or two) orders of magnitude larger than the decay time τ of the SiO surface. This work facilitates an understanding of the triboelectric and de-electrification of the interface between 2D materials and substrates. In addition to the charge transfer and diffusion, we demonstrate the nanopatterns of surface and interfacial charges, which have great potential for the application of self-assembly of charged nanostructures.