Objective To explore a method for instantly constructed TEB to meet the instantly clinical requirements of repairing bone defects. Methods BMNCs of Beagle were separated by 1.063 g/mL Percoll gradient solution. The ideal seeding density of 30 ×106 cells/mL was obtained for canine bone marrow mononuclear cells (cBMNCs) on partially demineralized bone matrix (pDBM). The cell activity and osteogenic capacity of immediately constructed TEB composed of cBMNCs and pDBM were analyzed in vitro. Results Certain cell activity and osteogenic capacity were confirmed in immediately constructed TEB under the osteogenically inducted environment in vitro. Conclusion The directly replantation of instantly constructed TEB might satisfy the immediately clinical requirements of repairing bone defects.