The field of tissue engineering, regenerative medicine, and in vitro models is undergoing transformative advancements, driven by the urgent need for structures that mimic the complex architecture and functionality of native tissues. Hierarchical scaffolds, characterized by macro-, micro-, and nanoscale features, have emerged as pivotal tools for efficiently regenerate different tissues/organs. These scaffolds can replicate the extracellular matrix with remarkable precision, offering structural support, enhanced cell alignment, and biocompatibility essential for effective tissue regeneration and in vitro models. Herein, the current advances in the biofabrication of hierarchical scaffolds, emphasizing the integration of advanced techniques such as additive manufacturing, electrospinning, freeze-drying, sol-gel, and self-assembly, are discussed. These methods enable the precise design of scaffolds tailored for diverse applications, from bone regeneration to disease modeling. The discussion extends to material innovation and selection criteria, balancing natural and synthetic polymers for optimized biocompatibility, mechanical stability, and controlled degradation. Additionally, sustainability is highlighted, showcasing renewable materials, energy-efficient processes, and circular economy principles that align with environmental responsibility. Hierarchical scaffolds offer transformative potential in regenerative medicine and in vitro three-dimensional (3D) models, overcoming challenges like vascularization and scalability. By harmonizing biofabrication, functionality, and sustainability, they pave the way for next-generation diagnostics and advanced in vitro 3D testing platforms to revolutionize healthcare and biomedical research.