Aluminum phthalocyanine nanoparticles (NP AlPc) possess the features that make them a promising photosensitizer. In particular, AlPc NPs do not fluoresce in free nanoform, fluoresce weakly in normal tissue, strongly in tumors and very strongly in macrophages. Also, such particles fluoresce and become phototoxic when contacting certain biocomponents. The type of biocomponents that bind to AlPc NPS defines intensity, lifetime, and spectral distribution of the fluorescence. This study aimed to investigate the peculiarities of nanophotosensitizer capturing in 3D models of cell cultures. The data obtained demonstrate that AlPc NPs are captured by cells inside the spheroid in the course of the first hour, as the fluorescent signal's growth shows. Having analyzed the fluctuations of the fluorescence signal of AlPc NPs inside a spheroid, we have also discovered that the cellular 3D models are heterogeneous. Laser irradiation (two-photon excitation at λ = 780/390 nm) resulted in photobleaching of fluorescence, which is probably associated with AlPc NP deactivation. Thus, the created model comprised of a 3D cell culture and AlPc NPs provides a better insight into metabolic processes in cells than monolayer 2D cell cultures. Besides, the model allows to evaluate the photodynamic effect depending on phenotypic properties of various areas in the heterogeneous 3D-structure.