In this paper, a series of Ni3Al films were successfully prepared by magnetron sputtering at different bias voltage, and then the bias voltage-dependent composition, deposition rate, microstructure, hardness and fracture toughness were studied in detail by XPS, XRD, SEM, AFM, nanoindentation and digital microhardness tester. The results showed that introducing the bias voltage could increase the kinetic energy of the ionized charged ions during sputtering, thus significantly improve the deposition rate, the density of the internal structure and the surface smoothness of Ni3Al films; In addition, the introduction of appropriate bias voltage could induce the formation of amorphous/nanocrystalline composite structure to improve the crystallinity of the films. The wrapped nanocrystalline composite with biphasic structure could provide a large number of grain boundaries, which strengthen the blocking effect on dislocations, leading to the dislocation stacking at the grain boundary and resulting in the increasement of hardness. Meanwhile, the existence of the large number of grain boundaries could also consume the energy of crack propagation to inhibit the generation of macrocracks, and significantly enhance the fracture toughness of Ni3Al films.