The condylar cartilage of the temporomandibular joint (TMJ) is connected to the subchondral bone by an osteochondral interface that transmits loads without causing fatigue damage. However, the microstructure, composition, and mechanical properties of this interface remain elusive. In this study, we found that structurally, a spatial gradient assembly of hydroxyapatite (HAP) particles exists in the osteochondral interface, with increasing volume of apatite crystals with depth and a tendency to form denser and stacked structures. Combined with nanoindentation, this complex assembly of nanoscale structures and components enhanced energy dissipation at the osteochondral interface, achieving a smooth stress transition between soft and hard tissues. This study comprehensively demonstrates the elemental composition and complex nanogradient spatial assembly of the osteochondral interface at the ultramicroscopic scale, providing a basis for exploring the construction of complex mechanical models of the interfacial region.