Transcription factors (TFs) are core players in the control of gene expression, evolutionarily designed to recognize a subset of specific DNA sequences and nucleate the recruitment of the molecular machinery responsible for gene transcription. How TFs assemble and move in the cell nucleus to locate and bind their DNA targets and cause a transcriptional response, remains mostly unclear. NF-Y is a highly conserved, heterotrimeric TF with important roles in both housekeeping and lineage-specific gene expression, behaving as a promoter organiser. Despite a large number of biochemical, structural and genomic studies of NF-Y, there is a dearth of experiments in single living cells; therefore, basic assumptions of NF-Y biology remain unproven in vivo. Here we apply dynamic fluorescence microscopy techniques (FLIM-FRET, RICS and FRAP) on different combinations of NF-Y subunits. We provide real-time quantitative measurements on NF-Y subunit association and diffusion kinetics in the nucleus. We show that NF-Y moves and binds chromatin as a trimeric complex in vivo. This study serves as a template to measure the dynamics of multimeric nuclear proteins, and sets the basis for increasingly complex questions that live cell microscopy will prompt in the near future in the field of gene regulation.