: Zircaloy coating is one of the crucial technical ways to improve the accident tolerance of nuclear fuel cladding, which enables the zirconium-water reaction problems to be solved. Zr2Al3C4 coating is one type of candidate solutions to improve the high-temperature oxidation resistance of zirconium claddings. However, little study has been performed on the synthesis of Zr2Al3C4 coatings on zirconium alloy substrates due to the inter-diffusion, as well as mismatches in the thermal expansion coefficients, between the Zr2Al3C4 coating and the substrates. In this study, Zr2Al3C4 coatings were prepared through room-temperature magnetron sputtering and post annealing on zirconium alloy (ZIRLO) subst rates with magnetron-sputtered Al/Mo-C interlayers. The effects of with or without Al/Mo-C interlayers on phase and microstructure of Zr-Al-C coatings after annealing were studied by XRD, SEM and TEM. It is found that the coatings with no interlayer are broken; and no Zr2Al3C4 phase was formed due to significant interdiffusion between the Zr-Al-C coating and the substrate during annealing at 800 ℃ for 3 h. The Al/Mo-C interlayers prevented elemental diffusion between Zr-Al-C coatings and substrates during a post-annealing process. The Al/Mo-C interlayers act as diffusion barriers and greatly reduce the stoichiometric deviations from Zr2Al3C4 phase, which facilitates the formation of the Zr2Al3C4 phase in the final coating. Moreover, this diffusion-barrier layers contribute to eliminating cracks induced by the difference of the thermal expansion coefficients exist between the Zr2Al3C4 coatings and substrates. At the same time, the adhesions between Zr-Al-C coatings with Al/Mo-C interlayers and substrates were improved after annealing, with their strength exceeds 30 N.