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A boronic acid groups-bearing polythiophene derivate (L) was designed and synthesized for highly sensitive fluorescence detection of ATP based on a multisite-binding coupled with analyte-induced aggregation strategy. L has a polythiophene backbone as fluorophores and two functional side groups, i.e., quaternary ammonium group and boronic acid group, as multibinding sites for ATP. When various structural analogues such as ADP, AMP, and various inorganic phosphates were added into the aqueous solution of L, only ATP caused a remarkable fluorescence quenching of about 60-fold accompanied by obvious color changes of solution from yellow to purple. The detection limit is estimated to be 2 nM based on 3σ/slope. With the advantage of good water solubility, low toxicity, and highly selective response to ATP, L was successfully utilized as a probe to real-time assay activity of adenylate kinase (ADK) and map fluorescent imaging of ATP in living cells.