The aim of this study was to improve the osteogenic ability of 3D-printed porous titanium (Ti) intervertebral spacer with a Tri-calcium Phosphate (TCP) loaded Demineralized Bone Matrix Hydrogel (DBM). Microstructures and morphologies of Ti, DBM loaded Ti (Ti/DBM), and TCP and DBM loaded Ti (Ti/DBM/TCP) scaffolds were characterized by SEM and EDX. In-vitro studies showed that Ti/DBM/TCP scaffold was biocompatible and could promote osteogenesis by up-regulating the expression of bone-related genes. Furthermore, the results of in-vivo studies using rabbit-femur defect model revealed that the implanted Ti/DBM/TCP scaffold had superior bone regeneration. The TCP and DBM loading were an effective approach of inducing osteogenesis in 3D-printed Ti intervertebral spacer by providing favorable osteogenic differentiation conditions and promoting bone formation.