Ultrasonic seam assisted resistance spot welding, as a new joining technology, was proposed for welding TC4 titanium alloy and Q235 mild steel with Cu interlayer (abbreviated as US-RSW-Cu), in which, the Cu interlayer was consolidated on the Q235 mild steel by ultrasonic seam welding (USW) in advance, which was subsequently connected with TC4 by the traditional standard resistance spot welding (RSW) in the following experiments. To investigate the effect of pre-USW on the interface microstructure and mechanical properties, the final joints were compared with the other joints by the traditional RSW without interlayer directly (abbreviated as RSW-D), and with a single Cu interlayer (abbreviated as RSW-Cu). The results depicted that the welding nugget in RSW-D and the flat interfacial structure in RSW-Cu were replaced by the inhomogenous interfacial structure in US-RSW-Cu due to the pre-metallurgical bonding between Cu interlayer and steel by USW, which also effectively decreased the welding defects. The Ti-Cu intermetallic compounds (IMCs) were generated at the final joints via US-RSW-Cu instead of most Ti-Fe IMCs, which improved significantly the joint properties and reduced the nanoindentation hardness at the joint interface. The final shearing force of welding joints by this method was the highest and reached 12,380 N, which was 2.68 times with original joint strength via a single RSW-D. The reaction layer near the Ti side via US-RSW-Cu contained abundant Fe atoms, forming Ti-Cu and TiFe2 IMCs at this position. The final welded specimen fractured at the interfacial reaction layer near the Ti side due to the generation of TiFe2 IMC. The XRD results also proved the generation of the abovementioned Ti-Cu and Ti-Fe IMCs.