Regioisomerism is an efficient strategy for finely tuning the structures of molecules in the isolated or the aggregated state. Herein, two regioisomers of perylene diimide (PDI) derivatives have been designed and synthesized. The positions of the substituent anthraquinone groups display distinct influences on their molecular conformation and aggregate structures, resulting in different fluorescence properties upon one- and two-photon excitation. In addition, owing to the bright emission, cellular imaging tests based on their nanoparticles were also conducted. The isomerism effect on the aggregate emission properties of perylene diimide derivatives is studied. Banking on their bright emission, a cellular imaging test based on their nanoparticles is also conducted.