Joining titanium alloy and stainless steel is becoming an urgent need since their outstanding mechanical properties can be utilized integratedly. However, direct fusion joining of Ti6Al4V to SS316 can cause brittle Ti-Fe intermetallics which compromise join bonds’ mechanical properties. In this research, Laser 3D Printing was applied to explore a new Ti6Al4V to SS316 multi-metallic structure. A novel filler transition route was introduced (Ti6Al4V → V → Cr → Fe → SS316) to avoid the Ti-Fe intermetallics. Two experimental cases were performed for comparison to evaluate this novel route’s effect. In the first case, SS316 layer was directly deposited on Ti6Al4V substrate by laser 3D printing, but the sample cracked in the printing process. Then fracture morphology, phase identification, and micro-hardness were analyzed. In the second case, a multi-metallic structure was fabricated via laser 3D printing following the transition route. Microstructure characterization and composition distribution were analyzed via scanning electron microscope(SEM) and energy dispersive spectrometry(EDS). x-ray diffraction(XRD) tests demonstrated the intermetallics were effectively avoided following the transition route. Vickers hardness number(VHN) showed no significant hard brittle phases in the sample. Comparing with directly depositing SS316 on Ti6Al4V, the usage of the novel transition route can eliminate the intermetallics effectively. These research results are good contributions in joining titanium alloy and stainless steel.