A pair of aza-BODIPY isomers, 1,7-di-tert-butyl-3,5-dinaphthyl (Nap-BDP) and 1,7-dinaphthyl-3,5-di-tert-butyl (revNap-BDP), were prepared in this study. According to the single crystal X-ray analysis, Nap-BDP exhibited an orthogonal structure. Owing to the difference in orthogonality and -tBu rotation between Nap-BDP and revNap-BDP, their spectral performances, including maximum absorption and emission, full width at half maximum, fluorescence quantum yield, photostability, singlet oxygen generation and photothermal conversion efficiency, were obviously different. The corresponding calculation results were in good agreement with the experimental results. Furthermore, the self-assembly of Nap-BDP nanoparticles could effectively promote cancer cell death by inducing singlet oxygen. Hence, this approach holds potential as a therapeutic strategy for enhancing cancer cell eradication via photodynamic therapy.