The effect of pyrenes introduced into a tobacco mosaic virus (TMV) coat protein monomer on the formation and stability of the TMV assembly was investigated. The possible arrangement of the pyrenes in the inner cavity of the TMV rod was also estimated. The pyrene derivative was introduced to four specific amino acids in the cavity of the TMV rod structure. Rod-structure formation was examined by atomic force microscopy (AFM). Two pyrene-attached mutants (positions 99 and 100) assembled to increase the length of the rod structures by 2.5 μm at pH 5.5. The interaction of the pyrene moieties in the TMV cavity was investigated by steady-state and time-resolved spectroscopic analysis. Strong excimer emission with significantly short wavelength (465 nm) was observed from the two mutants mentioned above. Excitation and UV-visible spectra indicate that the pyrene moieties form π-stacked structures in the TMV cavity. Details of the pyrene interaction were investigated by analyzing the fluorescence lifetime of the excimer. Results suggest that the pyrenes formed preassociated rigid structures with partially overlapped geometry in the restricted space of the TMV cavity. The pyrenes effectively stabilize the TMV rod through a π-stacking interaction in a well-ordered way, and the single pyrene moiety introduced into the monomer affects the overall formation of the TMV rod structure.