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Development of fluorescent imaging probes is an important topic of research for the early diagnosis of cancer. Based on the difference between the cellular environment of tumor cells and normal cells, several "smart" fluorescent probes have been developed. In this work, a glycopolymer functionalized Förster resonance energy transfer (FRET) based fluorescent sensor is developed, which can monitor the pH change in cellular system. One‐pot sequential reversible addition‐fragmentation chain transfer (RAFT)polymerization technique is employed to synthesize fluorescent active triblock glycopolymer that can undergo FRET change on the variation of pH. A FRET pair, fluorescein o‐acrylate (FA) and 7‐amino‐4‐methylcoumarin (AMC) is linked via a pH‐responsive polymer poly [2‐(diisopropylamino)ethyl methacrylate] (PDPAEMA), which can undergo reversible swelling/deswelling under acidic/neutral condition. The presence of glycopolymer segment provides stability, water solubility, and specificity toward cancer cells. The cellular FRET experiments on cancer cells (MDA MB 231) and normal cells (3T3 fibroblast cells) demonstrate that the material is capable of distinguishing cells as a function of pH change. [ABSTRACT FROM AUTHOR]