Welding is an essential means of joining structural components to form a new structure. Welding residual stress mainly results from materials expanding or contracting due to temperature variations, which can reduce the life of titanium alloys. Therefore, to reduce undesired residual stress, the welding process and microstructure of the materials involved should be optimized. Titanium alloys play a crucial role in marine and aviation fields due to their excellent corrosion resistance and high specific strength. This work investigates the influence mechanism of the prewelding pretreatment process on the structure, mechanical properties, and residual stress of the electron beam welding joint of a titanium alloy thick plate. The macrostructure and microstructure of titanium alloy welding joints prepared using different pretreatment processes are characterized. Results showed that preheating before welding substantially widens the fusion zone (FZ) and heat-affected zone (HAZ) of the welding joint, coarsening α lamellae in both zones. Thus, the hardness of the FZ and HAZ of the preheated welding joint is reduced to close to that of the base metal. Simultaneously, the strength and toughness of the welding joint is considerably improved such that it is similar to the base metal. The neutron diffraction, deep-hole drilling, and Rostenthal-Norton contour methods are used to measure the residual stress of the electron beam welding joint. The neutron diffraction method exhibits high detection accuracy and can achieve stress monitoring in different zones of the weld seam. Deep-hole drilling is a mechanical strain relief technique for measuring transverse and longitudinal residual stress through component thickness. The Rostenthal-Norton contour method can obtain a three-dimensional stress on the welding joint. A combination of these three measurement techniques can complement and be used to verify each other, providing reasonable data for the residual stress evaluation. The detection results of unpreheated welding joints are compared and analyzed, and the residual stress distribution in the FZ and HAZ zones along different directions is obtained. The FZ is subjected to tensile residual stress along all three directions. Alternatively, the HAZ is subjected to compressive stress along the transverse and longitudinal directions and tensile stress along the normal direction. The residual stress at base metal is small. Additionally, the residual stress results obtained by the deep-hole drilling method for the welding joints using two preheating processes are compared. The results showed that preheating before welding can considerably reduce residual stress at the weld. The reason is discussed in depth. Numerical simulation is used to calculate the changes in the temperature and stress fields under different preheating temperatures. The dynamic change rules of thermal stress under different preheating temperatures are obtained. Results showed that increasing the preheating temperature reduces thermal stress and the thermal expansion mismatch in different areas of the welded joint. Moreover, the microstructure, element distribution, and grain orientation of the FZ and HAZ of joints welded using two pretreatment processes are analyzed. Preheating coarsens the α lamellae and promotes the redistribution of alloy elements, thereby reducing the stress concentration between α and β phases. Besides, variant selection of the HAZ is induced by the preheating process. The number and differences in the orientation of α variants are decreased, thereby reducing the stress concentration between variants.