A dual photo- and pH-responsive spirooxazine-functionalized polymer was synthesized by functionalization of dextran with a spirooxazine derivative (SO-COOH). The functionalized dextran derivatives can form nanoparticles in aqueous medium. Under UV light irradiation, the spirooxazine-functionalized dextran (Dex-SO) nanoparticles isomerize to zwitterionic merocyanine-functionalized dextran (Dex-MC), which leads to aggregation. However, the process is reversible upon irradiation with visible light. Under acidic conditions, the hydrophobic spirooxazine is protonated, and the nanoparticles aggregate or swell at pH values of 5 or 3, respectively. The encapsulation of the hydrophobic fluorescent dye Nile Red as model drug allowed us to gain more information about the structural changes under stimulation of UV light and acid treatment.