Monodispersed diphenylalanine-based nanospheres with excellent biocompatibility are fabricated through a facile covalent reaction-induced assembly. Interestingly, the nanospheres exhibit red autofluorescence. Most importantly, such assembled dipeptide nanospheres can serve as intrinsic photosensitizer to convert O-2 to singlet oxygen (O-1(2)). Thus, photodynamic therapy in vitro can be achieved effectively. The versatile strategy could be extended to other biomolecules containing a primary amine group for the fabrication of potential intrinsic photosensitizers.