The mechanism of pathological osteogenesis in Ankylosing spondylitis (AS) is largely unknown. Our previous studies demonstrated that the imbalance between BMP-2 and Noggin secretion induces abnormal osteogenic differentiation of marrow-derived mesenchymal stem cells (MSCs) from AS patients in a two-dimensional culture environment. In this study, HA/β-TCP scaffolds were further used as a three-dimensional (3D) biomimetic culture system to mimic the bone microenvironment in vivo to determine the abnormal osteogenic differentiation of AS-MSCs. We demonstrated that when cultured in HA/β-TCP scaffolds, AS-MSCs had a stronger osteogenic differentiation capacity than that of MSCs from healthy donors (HD-MSCs) in vitro and in vivo. This dysfunction resulted from BMP2 overexpression in AS-MSCs, which excessively activated the Smad1/5/8 and ERK signalling pathways and finally led to enhanced osteogenic differentiation. Both the signalling pathway inhibitors and siRNAs inhibiting BMP2 expression could rectify the enhanced osteogenic differentiation of AS-MSCs. Furthermore, BMP2 expression in ossifying entheses was significantly higher in AS patients. In summary, our study demonstrated that AS-MSCs possess enhanced osteogenic differentiation in HA/β-TCP scaffolds as a 3D biomimetic microenvironment because of BMP2 overexpression, but not Noggin. These results provide insights into the mechanism of pathological osteogenesis, which can aid in the development of niche-targeting medications for AS.