The microstructure and high-cycle fatigue properties of 140-mm TC4 titanium alloy double-sided electron beam welded joints were studied by stratification along the thickness direction. It was found that the microstructure and properties of the weld zone were obviously inhomogeneity in the direction of thickness, and the overlapping area at the root of the two welds (the second layer of the joint) was the weakest position in the high-cycle fatigue performance of the joint. The larger hardness gradient was an important reason for its weaker than other regions. Microstructure differences was the primary reason for the high hardness of the second layer weld zone. It was found that the difference of heat dissipation conditions was a critical factor for the inhomogeneity of microstructure and mechanical properties along the thickness direction of 140-mm double-sided electron beam welding joint. The research results will provide data support for the application of double-sided electron beam welding technology in aerospace manufacturing.