Methylene blue (MB) is widely used as a photosensitizer in photodynamic therapy applications. However, it is easily reduced by reductases in biological environments, which hampers its further applications. Here, we developed a one-pot method to synthesize MB-encapsulated and poly(vinylpyrrolidone) (PVP)-modified zeolitic imidazolate framework-90 (ZIF-90) nanoparticles (MB@ZIF-90/PVP NPs). The NPs show intact crystalline structure with improved colloidal dispersity and stability both in water and in the medium for cell culture. The size of the enzymes is much larger than the pore size of ZIF-9; thus, the access of reductive enzymes to encapsulated MB is prohibited, resulting in the protection of MB's photodynamic activity. Furthermore, cell experiments confirm that MB@ZIF-90/PVP NPs have lower dark cytotoxity than equivalent free MB but can efficiently induce photodynamic damage to tumor cells even in the presence of reductive enzymes upon light irradiation.