The formation and evolution of microstructures at the Ni/Fe interface in dissimilar metal weld (DMW) between ferritic steel and austenitic stainless steel were investigated. Layered martensitic structures were noted at the nickel-based weld metal/12Cr2MoWVTiB steel interface after welding and post-weld heat treatment (PWHT). The formation of the interfacial martensite layer during welding was clarified and its evolution during PWHT was discussed by means of scanning electron microscopy (SEM), electron backscatter diffraction (EBSD), electron probe microanalysis (EPMA), focused ion beam (FIB), transmission electron microscopy (TEM), energy dispersive X-ray (EDX), transmission kikuchi diffraction (TKD), phase diagrams, and theoretical analysis. In as-welded DMW, the Ni/Fe interface structures consisted of the BCC quenched martensite layer and the FCC partially mixed zone (PMZ), which was the result of inhomogeneous solid phase transformation due to the chemical composition gradient. During the PWHT process, the BCC interfacial microstructure further evolved to a double-layered structure of tempered martensite and quenched martensite newly formed by local re-austenitization and austenite–martensite transformation. These types of martensitic structures induced inhomogeneous hardness distribution near the Ni/Fe interface, aggravating the mismatch of interfacial mechanical properties, which was a potential factor contributing to the degradation and failure of DMW.