Hyperbranched polymers (HBPs) are highly branched macromolecules with a three‐dimensional dendritic architecture. Due to their unique topological structure and interesting physical/chemical properties, HBPs have attracted wide attention from both academia and industry. In this paper, the recent developments in HBP self‐assembly and their biomedical applications have been comprehensively reviewed. Many delicate supramolecular structures from zero‐dimension (0D) to three‐dimension (3D), such as micelles, fibers, tubes, vesicles, membranes, large compound vesicles and physical gels, have been prepared through the solution or interfacial self‐assembly of amphiphilic HBPs. In addition, these supramolecular structures have shown promising applications in the biomedical areas including drug delivery, protein purification/detection/delivery, gene transfection, antibacterial/antifouling materials and cytomimetic chemistry. Such developments promote the interdiscipline researches among surpramolecular chemistry, biomedical chemistry, nano­technology and functional materials. Hyperbranched polymers have demonstrated great potential as excellent precursors in supramolecular self‐assembly, and many delicate supramolecular structures from zero‐dimension to three‐dimension have been prepared, such as micelles, fibers, tubes, vesicles, membranes, large compound vesicles and so on. They have displayed promising applications in the biomedical areas including drug delivery, protein purification/detection/delivery, gene transfection, antibacterial/antifouling materials and cytomimetic chemistry.