In this paper, according to the structural characteristics of 140mm thick hollowed-out load-bearing TC4 titanium alloy beam, a double-side and double-pass electron beam welding (EBW) scheme with unequal penetration was proposed. The welding test was carried out and the microstructure and properties of the obtained joint were analyzed. It is found that the microstructure of the weld zone is obviously non-uniform in the direction of thickness, and the overlapping zone at the root of the two welds is the most noteworthy part. There were a lot of needle α′ phase and scattered β phase in the overlapping area of the root of the two welds, while the needle phase was short and the interlacing phenomenon of α′ phase was obvious. The needle α′ phase andα phase distributed in the original β grain boundary were observed in other weld areas of the joint, but the laths were longer and arranged in a more obvious direction. The microhardness of the weld in the overlap zone of the root of the two welds was about 10% higher than that of the other weld zones in the joint (341HV~346HV). At room temperature, the impact energy of the weld in the overlap zone at the root of the two welds was about 11% lower than that of the other weld zones in the joint (about 47J). The area where the roots of the two welds overlap was the area where the joint had the lowest tensile strength. With the aid of finite element simulation, it was found that the cooling rate around 900℃ of the overlapping area of the root of the two welds was higher than that of the other areas of the welds, which was an important reason for the difference in the microstructure and performance between the overlapping area and the other areas of the welds. It was noted that all joints were broken in the base material area during tensile tests, and it was believed that the inhomogeneity of microstructure and properties of large-thickness forgings in the direction of thickness resulted in the inhomogeneity of tensile strength of joints. The results show that it is feasible to use double-side and double-pass unequal penetration EBW welding scheme to fabricate thick hollowed-out load-bearing TC4 titanium alloy beam. Meanwhile, some beneficial measures such as improving the uniformity of microstructure and property of the base material and strictly controlling the interval time between two passes of welding are recommended