The aim of the work was to review the literature data regarding the prospects for the creation and use of multifunctional fluorescent two-dye nanosystems, which enable investigating the distribution of fluorescent components with significant acceleration of the study and introduction of nanomedicines into practice. Special attention is paid to the use of two substances with hydrophobic and hydrophilic properties in one nanoparticle (NP), capable of penetrating a living cell. The method of fluorescence confocal microscopy enables observation of the nanoscale dynamics of distribution and stability of drugs over time. The concomitant use of doxorubicin (DOX) and curcumin (CUR) in single nanoparticle causes synergism in the action of medical drugs, and their own fluorescence makes it possible to use them as multifunctional fluorescent nanosystems. Results. Data from the literature indicate that the use of two or more fluorescent dyes provide an advantage over other, more expensive methods when studying the penetration and distribution of NPs in living samples. The use of nanocarriers is an effective way to significantly increase the bioavailability of those drugs, which are poorly soluble in water. A promising direction of nanomedicine is the creation of complex bio-compatible multifunctional nanomaterials based on several active drugs, with the simultaneous use of their enhancers and the strategy of active targeting. Such recent structures enable targeted and controlled penetration of medicinal compounds into the sites of localization of pathological processes, reducing the toxicity of drugs to normal cells. Conclusions. The use of the fluorescence microscopy method, as exemplified by the two dyes, DOX and CUR, enables to trace the stages of interaction of loaded DOX and CUR nanoparticles with cultured cells, and their release from NPs to determine their amount and localization in organelles cells.