Micro/nanotopographical modification of biomaterials plays an essential role in the improvement of implants' cytocompatibility. In this paper, hierarchical micro/nanostructured surface was constructed to improve the titanium alloy cytocompatibility, which combined sandblasting, acid etching, and alkali heat treatment. The surface features were characterized using scanning electron microscope (SEM), laser scanning microscope, X-ray diffraction (XRD), and contact angle goniometer. A series of biological tests were carried out to evaluate cytocompatibility of bio-titanium alloy in vitro, including cell adhesion, counting, proliferation, osteocalcin expression, alkaline phosphatase (ALP) activity, and extracellular matrix mineralization. The results show that bio-titanium alloy with micro/nanostructured surface provided a beneficial interface which could promote the growth of osteoblasts. A uniform nanowire layer with high hydrophilicity was superposed on microstructures. Meanwhile, the biological tests indicate that hierarchical micro/nanostructures with micropits and nanowires could significantly promote osteoblasts' initial adhesion, proliferation, and differentiation. Moreover, the underlying mechanism of interaction between implants and cells was also discussed. This work proposes an effective approach to fabricate micro/nanostructured surface on titanium alloy for cytocompatibility improvement.