A conventional atomic force microscope (AFM) has been successfully applied to manipulating nanoparticles (zero-dimensional), nanowires (one-dimensional) or nanotubes (one- or two-dimensional) by widely used pushing or pulling operations on a single surface. However, pick-and-place nanomanipulation in air is still a challenge. In this research, a modified AFM, called a three-dimensional (3D) manipulation force microscope (3DMFM), was developed to realize 3D nanomanipulation in air. This system consists of two individually actuated cantilevers with protruding tips that are facing each other, constructing a nanotweezer for the pick-and-place nanomanipulation. Before manipulation, one of the cantilevers is employed to position nano-objects and locate the tip of the other cantilever by image scanning. During the manipulation, these two cantilevers work collaboratively as a nanotweezer to grasp, transport and place the nano-objects with real-time force sensing. The manipulation capabilities of the nanotweezer were demonstrated by grabbing and manipulating silicon nanowires to build 3D nanowire crosses. 3D nanomanipulation and nanoassembly performed in air could become feasible through this newly developed 3DMFM.