Disclosed in the present invention is a separation method for diamond layer. The method comprises the following steps: performing by using laser a two-dimensional scanning to an inner side of to be treated diamond, and forming a non-diamond layer in a depth below the to be treated diamond surface; removing the non-diamond layer to realize up-down separation of the diamond. The method will not damage surface of the diamond substrate, and compared to laser cutting technology, losses in the diamond cutting is reduced; and compared to the ion implantation separation method, cost is saved and manufacturing time is shortened.