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 the difference of 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) substrates with magnetron-sputtered Al/Mo-C interlayers. The effects of Al/Mo-C interlayers on phases and microstructures of Zr-Al-C coatings after annealing were studied by different methods. It is found that the coatings without interlayer are broken and no Zr2Al3C4 phase is formed due to significant interdiffusion between the Zr-Al-C coating and the substrate during annealing at 800 degrees C for 3 h. The Al/Mo-C interlayers prevented elemental diffusion between Zr-Al-C coatings and substrates during the 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 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 exceeding 30 N.