Gene expression and cell fate determination require precise and coordinated epigenetic regulation. The complex three-dimensional (3D) genome organization plays a critical role in transcription in myriad biological processes. A wide range of architectural features of the 3D genome, including chromatin loops, topologically associated domains (TADs), chromatin compartments, and phase separation, together regulate the chromatin state and transcriptional activity at multiple levels. With the help of 3D genome informatics, recent biochemistry and imaging approaches based on different strategies have revealed functional interactions among biomacromolecules, even at the single-cell level. Here, we review the occurrence, mechanistic basis, and functional implications of dynamic genome organization, and outline recent experimental and computational approaches for profiling multiscale genome architecture to provide robust tools for studying the 3D genome.