In response to the requirements for the long-term service reliability of Ni-based superalloys, the correlations between the microstructure and creep property are investigated in present work. Creep experiments were conducted on a designed Ni-based single crystal superalloy at 900 °C/330 MPa, the results indicate that the creep rupture properties deteriorated following thermal exposure at both 900 °C and 1000 °C. The γ′-phase, γ-matrix and γ/γ′ interface undergo varying degrees of degradation, including the coarsening and the decrease in the volume fraction of γ′-phase, the decrease in the γ-matrix strength, the decrease in the lattice misfit and the increase in the dislocation network spacing. When the values of the microstructural parameters (γ′-phase size and volume fraction) were numerically similar for different thermal exposure times at 900 °C and 1000 °C, taking 900 °C/10,000h and 1000 °C/2000h samples as examples. The experimental alloy exhibited better creep resistance after being exposed to 1000 °C compared to 900 °C. Through the combination of atom probe tomography and first-principles calculations, an interface strengthening mechanism driven by interface segregation is identified. Finally, a design concept for high-strength metal alloys based on the decoration of solutes interface segregation is proposed, also called “Interface Segregation Manipulation”.