Laser welding of Ti6Al4V and 316L stainless steel is performed using single Cu interlayer and composite Cu-Nb interlayer. The microstructural characterization and mechanical properties of Ti6Al4V/316L steel laser welded joints at various laser offsets were investigated. Proper laser offset distance to 316L steel side ensured weld integrity and restrained mass production of intermetallic compounds for single Cu interlayer. Energy dispersive spectroscopy showed that Cu solid solution and discontinuous Ti-Fe and Ti-Cu intermetallics were formed due to the dissolution and diffusion of atoms. The joint had a lower tensile strength and fractured along Ti6Al4V/Cu weld interface with obvious cleavage steps and tongue pattern. The inserting of Nb interlayer with high-melting point was conducive to prevent atomic diffusion and suppress the formation of brittle intermetallics. Similarly, the laser energy distribution was crucial to the weld formation and metallurgical reaction in fusion zone. The Cu sheet and 316L steel was mainly connected by heat conduction. The weld fusion zone consisted of (Cu, Nb) and (Ti, Nb) solid solution with no intermetallics. Compared to the welded joints with single Cu interlayer, the microhardness of fusion zone was reduced and tensile strength was improved because no brittle IMCs were formed with composite Cu-Nb interlayer. The maximum tensile strength reached 215 MPa and ductile fracture mode occurred with composite Cu-Nb interlayer, which was approximately 160% greater than that found with single Cu interlayer.