The development of cyclodextrin nanoassemblies as useful carriers for photosensitizer drugs (PS) delivery in biological environment is a topic of increasing interest. In this paper, we present a spectroscopic investigation on a nanosystem based on an amphiphilic cationic β-cyclodextrin derivative (CD-N) and an anionic porphyrin (TPPS). Nanoassemblies were prepared by hydration of an organic film containing the two species. The system was characterized by complementary techniques such as UV-vis, stationary and time-resolved fluorescence, and Dynamic Light Scattering (DLS) at different TPPS/CD-N molar ratios. Time-resolved fluorescence data showed that, at all the investigated molar ratios, TPPS is present both as self-aggregated species and monomers forming supramolecular adducts with CD-N. Moreover, DLS measurements evidenced families of aggregates having hydrodynamic radii ranging between 50 and 350 nm and the size distribution profile depending on the TPPS/CD-N molar ratio. At the highest CD-N concentration, the hydrodynamic radii of the aggregates were nearly the same as those of neat CD-N in the absence of TPPS (50 nm). No aging phenomena were registered, pointing out the high stability of these nanoassemblies in aqueous solution for at least a month. Preliminary studies on the internalization in tumoral cells and subsequent irradiation for PDT application were carried out. The results support the feasibility of these nanoaggregates to promote PS internalization in HeLa cells, inducing cell death upon visible light irradiation.