The distributions of tungsten (W) in Ni-based single crystal superalloys with different Re and Ta contents are characterized and specified by atom probe tomography. The results show that Ta changes the partitioning behavior of W and rejects it from the γ′-precipitate to the γ-matrix, while Re shows the opposite effect, which is consistent with the simulations via Thermo-Calc. Moreover, significant behaviors of W segregation at γ/γ′ interface are found in 2Ta2Re, 5Ta2Re, and 5Ta0Re alloys, contributing to the decrease in the interfacial energy. W interfacial segregations are conducive to decrease the interfacial energy. By comparing alloys with different W contents, W segregation is irrelevant to the W content, but highly W-partitioning-dependent. Additionally, nano-indentation tests also show that the addition of Re can significantly increase the hardness of γ-matrix, while Ta is conducive to strengthen within both γ-matrix as well as γ′-precipitates which is clarified by the solid solution strengthening calculations.