The effects of pre-strain treatment (10%–50%) on microstructure and tensile properties of a novel Ni-based superalloy at high temperature (600 ℃) were studied by EBSD, SEM and TEM. The results show that a large number of dislocation and stratification substructures are prefabricated in the alloy, and the strength of the alloy at 600 ℃ increases monotonically with the increase in the pre-deformation amount, while the elongation of the alloy decreases first and then increases. At the same time, when the pre-deformation amount is 50%, the yield strength of the alloy sample increases to 1516 MPa, the ultimate tensile strength is 1677 MPa, and the elongation after fracture is 2.47%. In addition, the strength improvement is mainly attributed to the L-C lock interaction formed by the intersection of the dislocation with the γ′ phase, the stacking fault, and the lamination fault on different (111) planes, which provides a new strengthening method for the regulation of high performance Ni-based superalloys.