Tetrapod zinc oxide whiskers (TZnO-Ws) were successfully prepared by a thermal oxidation method and confirmed using scanning electron microscopy (SEM) and wide angle X-ray diffraction (WAXD). A series of polyimide (PI)/TZnO-W composite films containing the as-prepared various TZnO-W contents were prepared and their physical properties were investigated to understand their potential use in flexible displays. The morphology, physical, and moisture barrier properties of the PI/TZnO-W composite films were interpreted as a function of the TZnO-W content. The PI/TZnO-W composite films exhibited an optical transparency greater than 80% at 550 nm (≤0.5 wt% TZnO-W content), a low coefficient of thermal expansion (CTE), and enhanced glass transition temperature. However, the thermal decomposition temperature decreased as the TZnO-W content increased. Although the mechanical strength increased up to 0.3 wt% TZnO-W content, it decreased due to poor interfacial interaction with high TZnO-W loading. The water vapor transmission rate (WVTR) and water uptake of the composite films, which were strongly dependent upon their morphological and chemical structure, varied from 508.3 to 325.2 g/m2/day and 3.7 to 1.3 wt%, respectively, greatly decreased as the TZnO-W content increased. The water resistance capacity of PI was greatly enhanced and moisture diffusion in the pure PI was retarded by incorporating the TZnO-W.