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Enzyme-regulated biomineralization offers precise spatiotemporal control over tissue mineralization, overcoming key limitations of conventional regenerative therapies. This review systematically examines the underlying biological mechanisms, focusing on enzymatic regulation of phosphate metabolism, mineralization regulators, and matrix stabilization that orchestrate hierarchical mineral deposition. Organic matrices facilitate nanoconfinement-driven nucleation and spatially controlled mineralization through biochemical functionalization. These fundamental mechanisms have inspired the development of advanced enzyme-functionalized biomaterials, such as covalently immobilized hydrogels, physically entrapped nanocomposites, bioaffinity scaffolds, and stimuli-responsive 3D-printed constructs, which enable precisely tunable in situ mineralization. In clinical applications, such biomaterial systems demonstrate significant therapeutic potential, with critical-sized bone defects showing accelerated healing through biomimetic mineral-collagen alignment and enzyme-mediated enamel restoration achieving both hardness recovery and reduced secondary caries incidence. Current limitations primarily involve enzymatic stability, immunogenicity, and manufacturing scalability. Emerging solutions focus on gene-enzyme hybrid platforms and intelligent responsive systems for personalized regenerative approaches. The synergistic integration of biological principles with materials science provides a transformative foundation for developing next-generation therapeutic strategies. Schematic illustration of enzyme-mediated mineralization processes. This diagram outlines key aspects of enzyme-regulated biomineralization, including immobilization strategies (physical encapsulation, covalent conjugation, and advanced carrier systems), the physiological roles of mineralization-related enzymes, and the development of enzyme-functionalized biomaterials such as engineered scaffolds, hydrogels, and 3D-bioprinted constructs. This integrated approach synergistically combines biochemical regulation with material design to facilitate biomimetic tissue repair and regeneration in bone and dental applications [Display omitted] •Enzymatic mineralization provides precise control, overcoming the limits of traditional grafts for tissue regeneration.•Enzyme-based biomaterials enable tunable mineralization, enhancing bone and dental regeneration via biomimetic alignment.•Stability and scalability challenges drive innovation toward smart, gene-enzyme hybrid platforms for personalized therapies.