In this work, a Ni2Al3 intermetallic compound coating was prepared on 45 # steel by combining methods of low-pressure cold spray and heat treatment. Firstly, powders mixed with Ni powders, Al powders, and Al2O3 powders as a mass ratio of 20:6:4 were sprayed on surface of 45 # steel by low-pressure cold spraying technology to prepare a Ni-Al pre-coating. Subsequently, the pre-coating was annealed at 570 °C in an argon atmosphere for 12 h to obtain the Ni2Al3 intermetallic compound coating. The composite coating was characterized using TEM, SEM, and XRD. High temperature oxidation performance of the composite coating was analyzed by isothermal oxidation tests conducted at 600 °C in air atmosphere for 96 h. The results show that the composite coating is composed of Ni2Al3 phase. Under a high-temperature oxidation environment, a protective oxide film composed by Al2O3 and NiO was formed on the coating surface, which resulting in a superior high-temperature oxidation resistance compared to 15CrMo heat-resistant steel. The mechanism of the Ni2Al3 coating resistance to high-temperature oxidation is that an oxide film mainly composed of Al2O3 and NiO formed on the surface during the high-temperature oxidation process; this oxide film can effectively resist oxidation and protect the substrate material from oxidation at a high temperature of 600 °C.