The technology trends of next generation electronic packaging are moving toward heterogeneous 3D packaging systems. One of the key processes of 3D packaging system is Cu-to-Cu bonding, which is highly dependent on the planarized, activated, and oxygen-free Cu surface. A two-step plasma treatment is studied to form a Cu surface that does not react with oxygen and improves the Cu bonding interface quality at low bonding temperature (300 °C). In this study, the effects of two-step plasma treatment on both sputtered and electroplated Cu surfaces were evaluated through structural, chemical, and electrical analysis. The Cu bonding interface was studied by scanning acoustic tomography analysis after the thermocompression bonding process. Both sputtered and electroplated Cu thin films had the preferred orientation of (111) plane, but sputtered Cu exhibited larger grains than the electroplated Cu. As a result, the roughness of sputtered Cu was lower, and the resistivity was higher than that of electroplated Cu. Based on X-ray photoelectron spectroscopy analysis, the sputtered Cu formed more copper nitrides and fewer copper oxides than the electroplated Cu. A significant improvement in bonding quality at the Cu bonded interface was observed in sputtered Cu.