Titanium is a biomaterial that meets a number of important requirements, including excellent mechanical and chemical properties, but has low bioactivity. To improve cellular response onto titanium surfaces and hence its osseointegration, the titanium surface was bio-functionalized to mimic an extracellular matrix (ECM)-like microenvironment that positively influences the behavior of stem cells. In this respect, fibronectin and elastin are important components of the ECM that regulate stem cell differentiation by supporting the biological microenvironment. However, each native ECM is unsuitable due to its high production cost and immunogenicity. To overcome these problems, a recombinant chimeric fibronectin type III9-10 and elastin-like peptide fragments (FN9-10ELP) was developed herein and applied to the bio-functionalized of the titanium surface. An evaluation of the biological activity and cellular responses with respect to bone regeneration indicated a 4-week sustainability on the FN9-10ELP functionalized titanium surface without an initial burst effect. In particular, the adhesion and proliferation of human mesenchymal stem cells (hMSCs) was significantly increased on the FN9-10ELP coated titanium compared to that observed on the non-coated titanium. The FN9-10ELP coated titanium induced osteogenic differentiation such as the alkaline phosphatase (ALP) activity and mineralization activity. In addition, expressions of osteogenesis-related genes such as a collagen type I (Col I), Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), osteocalcin (OCN), bone sialo protein (BSP), and PDZ-binding motif (TAZ) were further increased. Thus, in vitro the FN9-10ELP functionalization titanium not only sustained bioactivity but also induced osteogenic differentiation of hMSCs to improve bone regeneration.