Stem cell-based bone tissue engineering based on the combination of a scaffold and expanded autologous mesenchymal stem cells (MSCs) represents the current state-of-the-art treatment for bone defects and fractures. However, the procedure of such construct preparation requires extensive ex vivo manipulation of patient's cells to achieve enough stem cells. Therefore, it is impractical and not cost-effective compared to other therapeutic interventions. For these reasons, a more practical strategy circumventing any ex vivo manipulation and an additional surgery for the patient would be advantageous. Intraoperative concept-based bone tissue engineering, where constructs are prepared with easily accessible autologous cells within the same surgical procedure, allows for such a simplification. In this study, we discuss the concept of intraoperative construct preparation for bone tissue engineering and summarize the available cellular options for intraoperative preparation. Furthermore, we propose methods to prepare intraoperative constructs, and review data of currently available preclinical and clinical studies using intraoperatively prepared constructs for bone regenerative applications. We identify several obstacles hampering the application of this emerging approach and highlight perspectives of technological innovations to advance the future developments of intraoperative construct preparation.