Pyrene is a fluorescent spatially sensitive probe, with the unique ability to form excimers (excited-state dimers) by stacking interaction of two close molecules. Excimers have a characteristic emission spectrum, very different from the pyrene-monomer spectrum, ideal for conformational changes studies on proteins. A new engineered ferritin from archaea bacteria (HumAfFt) has been recently demonstrated to maintain the unique oligomerization properties catalysed by divalent cations of Archaeoglobus fulgidus ferritin at neutral pH. The association/dissociation equilibrium of HumAfFt from dimer to a cage-like 24-mer has been investigated exploiting pyrene fluorescence properties. A cysteine residue per ferritin monomer has been specifically introduced by mutagenesis inside the protein cage and the thiol moieties have been selectively labelled with N-(1-Pyrenyl)maleimide. Dramatic changes in the fluorescence occurred in the pyrene-labelled protein as the ferritin associate in its 24-mer state. Interestingly, the amount of excimer decreased with the assembly of the 24-mer. The kinetic of the process has been studied by stopped-flow measurements, highlighting a very fast association rate and a slightly slower dissociation rate, within the range of micro seconds. Pyrene excimer emission has also been measured by two-photon fluorescence microscopy by excitation in the near IR region, a low-energy excitation wavelength which can significantly reduce photodamage and photobleach in vitro experiments. These features, together with pyrene high stability and long fluorescence lifetime highlighted the potential of these new pyrene labelled probes.