Experiments of electron beam welding was carried out for a 50 mm thick TC4-DT titanium alloy. The effect of electron beam welding on the microstructure characteristics was investigated using optical microscope observation and microhardness measurement for the cross-section of the welded joints. The results show that the microstructure of base metal was typical bimodal structure composed of primary α phase and lamellar (α+β). The microstructure of weld metal was basket-like martensite α′. Coursed primary β grain boundaries were clear in upper and middle parts of weld metal, while the size of the β grains was small and the grain boundaries were not obvious in the bottom. The microstructure of heat affected zone (HAZ) may be divided into two regions. The microstructure of HAZ near fusion zone was consisted of little primary α phase and needle martensite α′, while the microstructure near base metal was constituted of primary α phase and transferred β containing aciculate α. The boundary of the two regions was dependent on the β phase transfer temperature during weld cooling. The microhardness of the weld metal and HAZ was higher than that of base metal, and there was a peak value of microhardness in the HAZ near fusion zone. In addition, the microhardness of the fusion zone had an increased tendency with the measured weld depth increasing