In the present work, low cycle fatigue (LCF) behavior of Ti-35421 titanium alloy with bimodal microstructure consists of lath α (αp) and βtrans were investigated by strain-controlled mode at room temperature. Results indicated that the cyclic stress amplitude of the bimodal microstructure Ti-35421 alloy show cyclic softening at first, then reach to cyclic stability at high strain amplitude (Δεt/2=1.0%, 1.2%, 1.4%, 1.6%). However, the cyclic stress response was characterized by cyclic saturation at low strain amplitudes (Δεt/2=0.6%, 0.8%). One of fatigue crack source was found by fracture morphology observation when Δεt/2=0.6%, while a large number of small secondary cracks occurred on the surface. On the contrary, multiple fatigue crack sources generated when the strain amplitude increased to 1.6%, the number of secondary cracks reduced, but the length and width of the secondary cracks increased significantly. TEM results indicated that a large number of dislocations generate at the αp/βtrans interface at the low strain amplitude (Δεt/2=0.6%), which might lead to micro-crack nucleation due to the stress concentration. Meanwhile, at high strain amplitude ( Δεt/2=1.6%), deformation inhomogeneity phenomena happened in the αp phase, a large number of dislocation tangles and dislocation debris formed in the αp phase, and some dislocation pile-ups formed in the αs phase, which is not founded in the β substrate.