In this study, special attention was paid to the correlation between the degree of ionization of the components and the effective formation of the complex under alkaline conditions. Using UV-Vis, 1H NMR, and CD, structural changes of the drug depending on the pH were monitored. In the pH range of 9.0 to 10.0, the G4.0 PAMAM dendrimer can bind 1 to 10 DOX molecules, while the efficiency increases with the concentration of the drug relative to the carrier. The binding efficiency was described by the parameters of loading content (LC = 4.80–39.20%) and encapsulation efficiency (EE = 17.21–40.16%), whose values increased twofold or even fourfold depending on the conditions. The highest efficiency was obtained for G4.0PAMAM-DOX at a molar ratio of 1:24. Nevertheless, regardless of the conditions, the DLS study indicates system aggregation. Changes in the zeta potential confirm the immobilization of an average of two drug molecules on the dendrimer’s surface. Circular dichroism spectra analysis shows a stable dendrimer-drug complex for all the systems obtained. Since the doxorubicin molecule can simultaneously act as a therapeutic and an imaging agent, the theranostic properties of the PAMAM-DOX system have been demonstrated by the high fluorescence intensity observable on fluorescence microscopy.