Titanium (Ti) is the most widely used orthopedic and dental implant material due to its excellent mechanical properties and biocompatibility. However, the bioinert nature of Ti often leads to prolonged osseointegration process, and even can result in implant failure. This study explored the use of femtosecond laser surface processing to create micro-nano pit structure on Ti surface, aiming to enhance the biological properties. The pit structure was characterized by SEM, AFM, XRD, XPS and contact angle meter. The biological performance of the laser-treated surface was evaluated through in vitro cell culture experiments. Results indicated that the femtosecond laser-induced micro-nano pit structure significantly improved cell adhesion, proliferation, and osteogenic differentiation compared to untreated and sandblasted Ti surfaces, and it also promoted the differentiation of mesenchymal stem cells (MSCs) into osteoblasts. The enhanced biological property is attributed to the formation of a micro-nanostructure and a bioactive oxide layer. This study indicates that femtosecond laser processing is a promising technique for improving the osseointegration of Ti-based implant materials.