AbstractLinear DNA undergoes a series of compression and folding events, forming various three‐dimensional (3D) structural units in mammalian cells, including chromosomal territory, compartment, topologically associating domain, and chromatin loop. These structures play crucial roles in regulating gene expression, cell differentiation, and disease progression. Deciphering the principles underlying 3D genome folding and the molecular mechanisms governing cell fate determination remains a challenge. With advancements in high‐throughput sequencing and imaging techniques, the hierarchical organization and functional roles of higher‐order chromatin structures have been gradually illuminated. This review systematically discussed the structural hierarchy of the 3D genome, the effects and mechanisms of cis‐regulatory elements interaction in the 3D genome for regulating spatiotemporally specific gene expression, the roles and mechanisms of dynamic changes in 3D chromatin conformation during embryonic development, and the pathological mechanisms of diseases such as congenital developmental abnormalities and cancer, which are attributed to alterations in 3D genome organization and aberrations in key structural proteins. Finally, prospects were made for the research about 3D genome structure, function, and genetic intervention, and the roles in disease development, prevention, and treatment, which may offer some clues for precise diagnosis and treatment of related diseases.