In this study, the relationship between different medium-temperature deformation heat treatment processes and the microstructure and room-temperature mechanical properties of a novel Ni-based superalloy was investigated using EBSD, SEM and quasi-static room-temperature uniaxial tensile tests. It is shown that the length fraction of annealed twins of the novel Ni-based superalloy can be significantly increased up to 40.6% after the medium temperature deformation heat treatment. The formation of annealed twins is mainly based on the "growth accident" mechanism of the grains. At the same time, compared with the mechanical properties of the solid solution + double-stage aging specimens (σ_y = 1018 MPa, ε_f = 17.44 %), the alloy annealed at 1120 °C for 30 min after 30 % roll deformation at 750 °C and double-aged, the σ_y increased by 499 MPa to 1517 MPa, while the ε_f decreased by only 4.69 %. After rolling at 750 °C for 50 % of the deformation and annealing at 1120 °C for 30 min with double-stage aging, the σ_y increases by 352 MPa to 1370 MPa, while the ε_f remains essentially unchanged. This increase in strength is mainly attributed to the combined effect of grain refinement and annealing twinning, which provides a new strengthening strategy for high performance Ni-based superalloys.