Manufacturing multi-material part is one of the native advantages of selective laser melting (SLM) due to its layer-by-layer manufacturing method, which is attracting more and more attention in recent years. In an effort to reveal the dual interfacial characterization of dissimilar materials manufactured by SLM with different printing sequences, this paper presents the morphology, microstructure, element distribution, phase composition and microhardness of multi-material interfaces between SLMed 316L steel and C52400 copper alloy. Both interfaces display isolated alloy islands with various shapes and different morphologies. The melt pool at 316L/C52400 interface is deeper and narrower than that at C52400/316L interface. Small 316L and C52400 spheres with a size of 1–5 μm are formed under the surface tension, Marangoni convection in the melt pool and rapid cooling condition, in which many smaller particles with a size of <1 μm appear due to the material supersaturation and convection. Interdiffusion of elements and very fine grains with a size of <6 μm result in excellent metallurgical bonding performance. Cracks tend to originate from the interface and extend to the stainless steel side for both interfaces. No separation of the two materials caused by cracks is found in their contact area. Healing of the cracks by C52400 copper alloy at 316L/C52400 interface is easier to complete. The interface thickness depends on the building sequence of the two materials, which features a much thicker transition when building C52400 on 316L. No intermetallic compound but a trace of CuNi alloy is formed at the interface. The microhardness varies in the transition area due to the existence of isolated 316L/C52400 islands and decreases from 316L to C52400 with the highest value of 283.33 ± 5.51 HV to the lowest value of 181.33 ± 17.62 HV. This research advances the understanding of the different interfacial characterizations of dissimilar materials manufactured by SLM and provides guidance and reference for manufacturing multi-material components with complex interfaces using SLM. •Multi-material interfaces of 316L stainless steel and C52400 copper alloy are manufactured with selective laser melting.•The interfaces display excellent metallurgical bonding performance with fine grains and interdiffusion of elements.•The 316L/C52400 interface has much wider transition zone than the C52400/316L interface.