Effective and fast osseointegration is important for the survival of titanium-based orthopaedic implants. Previous studies confirmed that topographic features combined with inorganic ions showed positive effects on the biological functions of osteoblastic cells. In this study, we report an approach for fabricating Fe-incorporated micro-nano hierarchical structures on titanium substrates, which was realized by dual acid etching and subsequent hydrothermal treatment. The surface morphology, surface chemistry and wettability of the titanium substrates were characterized using scanning electron microscopy, X-ray photoelectron spectroscopy and contact angle measurements, respectively. The Fe-incorporated micro-nano hierarchical titanium substrates were probed to be biocompatible and positively improved protein adsorption, cell proliferation and cell differentiation of osteoblasts in vitro. Furthermore, the Fe-incorporated titanium substrates significantly enhanced the expressions of osteogenic genes (such as Runx2, Col I, OPN, and OCN), which were attributed to the synergistic effects of micro-nano structures and Fe ions. More importantly, the Fe-incorporated titanium implants with micro-nano hierarchical structures promoted new bone formation in vivo. This study provides an alternative for the development of orthopaedic implants with improved osseointegration for potential clinical applications.