GH4068 was prepared by electron beam layer smelting (EBLS), and the effects of different layer thicknesses on the microstructure and element segregation of the samples were studied. The results show that the sample with a solidification thickness of 5.2 mm (533 g) has the most severe element volatilization. The overall structure was composed of coarse disordered grains at the bottom and continuous columnar grains at the bottom. A fine-grained layer appears between the sample layers with a solidification thickness of 7.8 mm, and the microhardness of the fine-grained layer can reach 452.2 HV. For the sample with a solidification thickness of 15.5 mm, the electron beam penetration capacity is insufficient due to the large thickness of the layer, which results in interlayer gaps. Under the premise of controlling the volatilization degree of the elements, the ingot segregation degree with a solidification thickness of 7.8 mm is the smallest, and the secondary dendrite spacing is the smallest. The most serious segregation elements are Ti and W. Compared with those of the traditional duplex smelting process, the W and Ti segregation degrees of the samples prepared by electron beam layer smelting are reduced by 11.2% and 6.65%, respectively. In summary, the optimal layer thickness is 7.8 mm.