The microstructure and deformation behavior around the fatigue crack tip can significantly affect the early stage of the fatigue crack propagation, but currently, the evolution and deformation mode of the microstructure in the fatigue crack tip at the nanoscale are still unclear. Therefore, it is important to study the microstructure and deformation behavior at the fatigue crack tip in nanoscale to further reveal the fatigue failure mode of metal materials. In this study, the microstructure evolution and deformation mode of the fatigue crack tip of nickel-based alloy are studied by HRTEM technique. The results show that a large number of dislocation, stacking fault and micro/nano-twin are formed around the fatigue crack tip. The formation of these microstructures can promote the release of strain in the matrix during cyclic deformation. The main deformation mode of the fatigue crack tip includes slip, twinning, and dislocation shearing micro/nano-twin.