In the present study, we report on the fabrication of novel amphiphilic reversible photoswitchable fluorescent nanoparticles by a facile one-pot miniemulsion polymerization using a biocompatible polymerizable nonionic surfactant, ω-methoxy poly(ethylene oxide) undecyl α-methacrylate (PEO-R-MA-40), in which spiropyran-linked methacrylate (SPMA) and the energy level well-matched fluorescent dyes: fluorescein-based, vinylic crosslinking monomer (fluorescein-O,O-bis-propene (FBP)) were covalently incorporated into polymeric matrix during the polymerization process. Under the alternative irradiation of UV and visible light, the fluorescent emission of FBP dye in nanoparticles can be reversibly modulated by cyclical transformation of spiropyran moieties' structure on the basis of intra-particle FRET process between FBP and merocyanine (MC) state of spiropyran. The as-prepared fluorescent nanoparticles display high dye load, as well as controllable amount and ratio of the two dyes because of covalent linkage between dye molecules and the particle. Moreover, the observed FRET efficiencies (66.3–98.5%) can be facilely tuned by controlling the amount (ratio) of the two chromophores. Overall, significant features of these novel amphiphilic reversible photoswitchable fluorescent nanoparticles include higher biocompatibility and photostability, relatively fast photoresponsivity and better photoreversibility as compared to some previous reported systems, which are promising for biological, optical fields and so on.