A comprehensive investigation of the surfaces of copper through silicon vias (Cu-TSVs) and gold stud bumps is presented. These vias and stud bumps were bonded at room temperature using a nanobonding and interconnection equipment. The influence of heating on the bonded interface was also studied. In order to achieve an intimate contact between the Au-stud bumps and Cu-TSVs, the stud bumps were flattened under an external force of 20 N before bonding. The surface roughness of the flattened area was improved due to deformation of the bumps. Specimens with high deformation provided better alignment accuracy than those with low deformation. The Cu-TSV surface showed inhomogeneous behavior due to the influence of electroplating and chemical mechanical polishing. Tensile pulling test of the bonded interfaces showed three fractures modes in the bulk of the Au bump and the Au pad. The electrical resistance of the bonded interface was dependent on the surface morphology of the bumps and TSVs, the distance between the bumps and TSVs, the locations of the bumps and TSV with respect to the argon fast atom beams, and the distribution of external force during bonding. Heating at 200 °C for 60 h in air increased the electrical resistance of the bonded interface. This investigation shows that the vertical integration of Au/Cu at room temperature and low bonding force can be applied to three-dimensional interconnections for low cost miniaturized systems.