In this work, interlayer (Cr vs amorphous CuZr) with various thicknesses ranging from 5 nm to 40 nm were utilized for 1000 nm-thick nanostructured Cu films on polyimide substrates. The effects of heterogeneous interface and interlayer thickness on the fatigue behavior of Cu/interlayer bilayers were investigated by in situ synchrotron X-ray diffraction, electrical resistance measurement, in combination with microstructural examinations. The results demonstrated that the fatigue tolerance highly depends on the thickness and constituent of interlayer. The fatigue lifetime monotonically increases as CuZr interlayer thickness increases, coupled to the yield stress. In contrast, the fatigue lifetime first increases and then decreases with increasing Cr interlayer thickness, reaching its maximum value at a thickness of 5 nm. The underlying mechanisms for this discrepancy can be elucidated in terms of heterogeneous constraint and interface voiding, both of which depend on the interlayer thickness.