Abstract Mesoscale building blocks are instrumental in bridging multilevel hierarchical mineralization, endowing macroscale entities with remarkable functionality and mechanical properties. However, the mechanism orchestrating the homogeneous morphology of mesoscale mineralized motifs in collagen-based hard tissues remains unknown. Here, utilizing avian tendons as a mineralization model, we reveal a robust correlation between the mesoscale mineralized spherules and the presence of phosvitin. By designing a phosvitin-stabilized biomineral cluster medium, we replicate the well-defined mesoscale spherical structure within collagen matrix in vitro and ex vivo. In-depth studies reveal that phosvitin undergoes a conformational transition in the presence of biominerals at physiological concentrations, and self-assembles into mineral-dense amyloid-like aggregates. The spatial binding of these mineral-dense aggregates to collagen serves as a template for guiding the formation of mineralized spherules on the mesoscale. On the nanoscale, this binding facilitates mineral precursor release and diffusion into the fibrils for intrafibrillar mineralization. This discovery underscores the pivotal role of phosvitin-biomineral aggregates in templating hierarchical mineralization from the mesoscale to the nanoscale. This study not only elucidates the intricate mechanism underlying the collagen-based mineralization hierarchy but also promotes a cutting-edge advance in highly biomimetic material design and regenerative medicine.