A nano-twinned copper layer is disclosed, wherein over 50% of a volume of the nano-twinned copper layer comprises a plurality of columnar crystal grains, the plurality of columnar crystal grains connect to each other, at least 70% of the plurality of columnar crystal grains are formed by a plurality of nano-twins stacking in an orientation of a crystal axis, and an angle included between two adjacent columnar crystal grains is greater 20° and less than or equal to 60°. In addition, a method for manufacturing the nano-twinned copper layer and a substrate comprising the same are also disclosed.