Multilayered structures of dissimilar titanium alloys can achieve excellent fracture ductility and strength, while their fatigue characteristics especially dislocation networks and twin formation are rarely reported. Heterogeneous microstructures are observed in the multilayered TC4/TB8 alloys, including fine acicular α grains, continuous α layer at prior β grain boundaries (αGB) and β matrix on the TB8 layer, together with equiaxed α grains on the TC4 layer. High-cycle fatigue (HCF) and low-cycle fatigue (LCF) tests show that the initial fatigue damage appears at the αGB/β matrix interfaces on the TB8 layer instead of the boned TC4/TB8 interfaces. Since the stress concentration induced by dislocation pile-up is prone to micro-void formation and crack propagation at the αGB/β interfaces. For LCF, the αGB/β interfaces can not only act as impenetrable barriers and sources of lattice dislocations, but also allow the dislocations cross boundaries during cyclic tension and compression because of the high boundary energy. The formation characteristic of deformation twins that is beneficial for the plastic deformation of α grains in TC4 layer during cyclic strain is investigated. Furthermore, the hexagonal dislocation networks are also found within the equiaxed α grains of TC4 layer after LCF, and the role between interface barrier and slip direction in the formation mechanism is analyzed.