The effects of post-weld heat treatment on the microstructure and mechanical properties of Ti650 alloy electron beam welding (EBW) samples were studied. The results indicated that metastable martensite α′ is the main phase in the fusion zone of welding seam. During 700℃/2hAC heat-treatment, a phase transition α′→α occurred, and a large number of short aciculate secondary α were precipitated in the fusion zone. After 1010℃/1.5hWC+650℃/2hAC heat treatment, α was obviously coarsened and equiaxed. The microstructure contains the original coarse layer and the secondary short acicular alpha can effectively improve the strength and prevent the crack growth of welded joint. The welded joint has better strength and plasticity under the condition. After subsequent treatment at 700℃/2hAC, some equiaxed alpha can be found gradually precipitated at the grain boundary, resulting in a decrease in strength and plasticity of the grain boundary. In conclusion, 1010℃/1.5hWC+650℃/2hAC is the most suitable post-welding heat treatment for Ti650 EBW welded joint to make the properties of weld and matrix be well matched.