A nano-twinned copper layer with a doped metal element is disclosed, wherein the nano-twinned copper is doped with at least one metal element selected from the group consisting of Ag, Ni, Al, Au, Pt, Mg, Ti, Zn, Pd, Mn and Cd in a region from a surface of the nano-twinned copper layer to a depth being 0.3 μm, and a content of the metal element in the region is ranged from 0.5 at % to 20 at %. In addition, at least 50% in volume of the nano-twinned copper layer includes plural twinned grains. Furthermore, a substrate including the aforesaid nano-twinned copper layer and a method for preparing the aforesaid nano-twinned copper layer are also disclosed.