Atoms, molecules, and nanoparticles can be spatially manipulated by an atomic force microscopy (AFM) tip, through van der Waals (vdW) and/or Coulomb forces. These point-to-point manipulations are highly accurate at nanoscale, facilitating the construction and modification of nanostructures. Nevertheless, it is difficult to manipulate 2D layers in vdW crystals by an AFM tip, because the tip-induced attractive force is usually insufficient to outcompete the interlaminar vdW forces. Herein, manipulation of the surface layers on a MoS2 single crystal by a conductive AFM tip is successfully reported. By applying a bias between the tip and MoS2, the Coulomb attractive force allows the topmost MoS2 layers to be picked up. These exfoliated layers are deformed into micron-sized bubbles with sixfold symmetry, which are composed of high-quality monolayers and visually reflecting the hexagonal lattice orientation. The underlying mechanisms of the sixfold symmetric exfoliation and the formation of monolayers are discussed by in situ monitoring of the tunneling volt-ampere characteristics and simulation of the force distribution. The findings open a new route to obtain high-quality transition metal dichalcogenide (TMD) monolayers and their derived nanostructures on the surface of TMD single crystals for optoelectronic and photonic device applications.