Interface plays an important role in determining several properties in multiphase systems. It is also essential for the accurate measurement of the interface structure in a single crystal Ni-based superalloy (SCNBS) under different conditions. In this work, a subpixel accuracy transform method is introduced in detail to measure SCNBS lattice spacing at high temperatures. An intensity ratio analysis based on a high-resolution transmission electron microscopy image is employed for SCNBS interface width analysis. In this particular sample, the interface width is about 2 nm. The evolution of the lattice spacing of an ordered γ' phase and a solid solution γ matrix is also obtained at high temperatures. The lattice misfit between the matrix γ phase and the γ' precipitation increases with the temperature, with values of -0.39% and -0.21% at 20°C and 600°C. In addition, the coefficient of the SCNBS thermal expansion at high temperatures is discussed.