Pi-stacked perylenediimides (PDIs) have strong electronic communication between the individual molecules and show great promise as organic electronic materials for applications in field effect transistors, photovoltaics, and liquid crystal displays. To gain further insight into the relationship between conformational behaviors and electronic structures of pi-stacked PDIs, we have investigated changes in the excimer-like state of cofacial PDI oligomers that result from pi-stacking in real time by monitoring the single-molecule fluorescence intensity and lifetime trajectories in a PMMA polymer matrix. The fluorescence intensity and lifetime of pi-stacked perylenediimides are sensitive to the degree of pi-orbital interactions among PDI units, which is strongly associated with molecular conformations in the polymer matrix. Furthermore, our results can be applied to probe the conformational motions of biomolecules such as proteins.