This paper reveals the evolution of interfacial microstructure and its effect on the mechanical properties of Al alloy/stainless steel joint made by inertia friction welding with different surface roughness (as-machined and polished) followed by preheat treatment. At the central interface region, an unbonded area is witnessed for the Al alloy/as-machined steel (AA-MS) joint, while an intimate contact is realised for the Al alloy/polished steel (AA-PS) joint. The 1/2 radius (1/2R) region is characterised by the formation of the Fe–Al reaction layer, and no obvious layer exists at the peripheral region. Further microstructural characterisation indicates the reaction layer consists of a partially nanoscale crystallised layer of Fe2Al5 and a nanoscale amorphous layer in the as-welded joint induced by the severe shear effect. Preheat treatment promotes the growth of the crystallised layer and suppresses the amorphous layer due to the enhanced atom mobility. Preheat treatment improves the local interfacial bonding strength by reducing the unbounded area while maintaining a high bonding quality at the 1/2R region in the AA-MS joint. However, the enlarged brittle peripheral region is responsible for a slightly lowered joint strength in the preheated AA-PS joint.