Tissue engineered bone grafts (TEBG) using mesenchymal stem cells (MSCs) demonstrate great potential for bone defect treatment. However, current MSC expansion techniques and multiple-step TEBG construction strategy have problems such as repeated trypsinization, limiting further clinical application. Microcarriers present promising solutions, but conventional microcarriers are either non-implantable or have insufficient biomimetic potential to maintain effective cellular function. Here, we developed a biomimetic and implantable microcarrier using demineralized bone matrix (DBM-MC), which preserves the essential biochemical composition, architecture and surface topography of natural bone tissue. Furthermore, based on this DBM-MC, we established a single-step micro-sized TEBG (μTEBG) construction strategy integrating multiple procedures of cell seeding, expansion, and differentiation. Benchmarked with Cytodex 3, a widely used microcarrier, DBM-MC shared similar physical properties, and supported efficient cell adhesion and proliferation with MSC characteristics being well maintained. However, when implanted ectopically, the MSC/DBM-MC constructs achieved more neo-bone formation with better vascularization than MSC/Cytodex 3. Moreover, μTEBG generated via our single-step strategy can successfully heal a critical-sized cranial defect with two-fold more bone regeneration. This new DBM-MC and single-step μTEBG construction strategy can provide an enclosed, large-scale, reduced-trypsinization, and semi-automatic fabrication process to generate μTEBGs with outstanding osteogenic and angiogenic capacity, demonstrating great potential for clinical application.