To analyze the effect of diffusion bonding process on vibration fatigue properties, a series of fatigue tests and microstructure characterization of as-received and diffusion bonded TC4 titanium alloy were conducted. The results show that the thermal cycling of diffusion bonding process results in microscopic change and deteriorates vibration fatigue lifetime of TC4 alloy. For bonded TC4 alloy, the increased grain size inhomogeneity stimulates incompatible deformation of differently oriented α grains and leads to micro-void formation at boundaries of small/large α grain pairs or inside large α grains. Meanwhile, the orientation variation of α grains promotes activation of prismatic slip systems for earlier crack initiation. In addition, the increased fraction of β phase provides more nucleation locations for micro-voids. Thus, the increase of the grain size inhomogeneity, grain orientation change, and the increase of β phase fraction due to the thermal cycling result in fatigue performance deterioration of diffusion bonded TC4 titanium alloy.