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Organic and polymer fluorescent nanomaterials are a frontier research focus. Here in this work, a series of fluorinated zwitterionic random copolymers end‐attached with a quasi‐chromophoric group of pyrene or tetraphenylethylene (TPE) are well synthesized via atom transfer radical polymerization with activators regenerated by electron transfer (ARGET ATRP). Those random copolymers with total degree of polymerization 100 or 200 are able to produce fluorescent single‐chain nanoparticles (SCNPs) through intra‐chain self‐folding assembly with quite uniform diameters in the range of 10–20 nm as characterized by dynamic light scattering and transmission electron microscopy. By virtue of the segregation or confinement effect, both SCNPs functionalized with pyrene or TPE group are capable of emitting fluorescence, with pyrene tethered SCNPs exhibiting stronger fluorescence emission reaching the highest quantum yield ≈20%. Moreover, such kind of fluorescent SCNPs manifest low cytotoxicity and good cell imaging performance for Hela cells. The creation of fluorescent SCNPs through covalently attached one quasi‐chromophore to the end of one fluorinated zwitterionic random copolymer provides an alternative strategy for preparing polymeric luminescence nanomaterials, promisingly serving as a new type of fluorescent nanoprobes for biological imaging applications. [ABSTRACT FROM AUTHOR]