Novel amphiphilic photoswitchable thiophene-based fluorescent polymer dots (Pdots) were fabricated by coprecipitating a soluble and highly fluorescent polythiophene precursor with another photochromic spiropyran-linked amphiphilic copolymer into water under continuous ultrasound. The designed spiropyran-linked amphiphilic copolymer not only provided the fluorescent Pdots with unprecedented long-term photostability (>30 days) in aqueous media, but also allowed the reversible modulation of the fluorescence emission of the thiophene-based polymer via alternating irradiation with UV and visible light. These fluorescent Pdots not only show a uniform small particle size (ca. 16 nm in diameter), excellent long-term stability and high brightness of single particles, but also reveal distinct dual-color fluorescence, fast photo-responsiveness, as well as favorable photoreversibility with a fatigue resistance over at least 10 cycles of photoconversion.