Cell fate changes during development, differentiation, and reprogramming are largely controlled at the transcription level. The DNA-binding transcription factors (TFs) often act in a combinatorial fashion to alter chromatin states and drive cell type-specific gene expression. Recent advances in fluorescent microscopy technologies have enabled direct visualization of biomolecules involved in the process of transcription and its regulatory events at the single-molecule level in living cells. Remarkably, imaging and tracking individual TF molecules at high temporal and spatial resolution revealed that they are highly dynamic in searching and binding cognate targets, rather than static and binding constantly. In combination with investigation using techniques from biochemistry, structure biology, genetics, and genomics, a more well-rounded view of transcription regulation is emerging. In this review, we briefly cover the technical aspects of live-cell single-molecule imaging and focus on the biological relevance and interpretation of the single-molecule dynamic features of transcription regulatory events observed in the native chromatin environment of living eukaryotic cells. We also discuss how these dynamic features might shed light on mechanistic understanding of transcription regulation.