The biological performance of Ti-6Al-4V implant is primarily determined by their surface properties. However, traditional surface modification methods, such as acid etching, hardly make improvement in their osseointegration ability and antibacterial capacity. In this study, we prepared a multi-scale composite structure coated with zinc oxide (ZnO) on Ti-6Al-4V implant by an innovative technology of two-step laser processing combined with solution-assistant. Compared with the acid etching method, the physicochemical properties of surface significantly improved. The in vitro results showed that the particular dimension of micro-nano structure and the multifaceted nature of ZnO synergistically affected MC3T3-E1 osteogenesis and bacterial activities: (1) The surface morphology showed a 'contact guidance' effect on cell arrangement, which was conducive to the adhesion of filopodia and cell spreading, and the osteogenesis level of MC3T3-E1 was enhanced due to the release of zinc ions (Zn2+); (2) the characterization of bacterial response revealed that periodic nanostructures and Zn2+ released could cause damage to the cell wall of E. coli and reduce the adhesion and aggregation of S. aureus. In conclusion, the modified surface showed a synergistic effect of physical topography and chemical composition, making this a promising method and providing new insight into bone defect repairment.