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Water-soluble conjugated polymers are attractive fluorescent materials for applications in chemical and biological sensing. The molecular wire effect of such polymers amplifies changes in the fluorescence signal, which can be used for detecting various analytes with high sensitivity. In this work, we report an efficient ratiometric fluorescent probe based on a water-soluble conjugated polymer that showed high sensitivity and selectivity toward adenosine 5?-triphosphate (ATP). The macromolecular probe consisted of a polyfluorene backbone doped with 5 mol % 1,4-dithienylbenzothiadiazole (DBT) modified by bis-imidazolium and oligo(ethylene glycol) moieties. Solutions of the polymer emitted purple fluorescence, which changed to red upon addition of ATP molecules. The addition of ATP caused the polymer to aggregate, which enhanced fluorescence resonance energy transfer efficiency from the fluorene segments to DBT units, leading to an increase in red emission. The ratio of the fluorescence at these different wavelengths (I655/I423) showed a strong dependence on the ATP concentration. PF-DBT-BIMEG also exhibited high selectivity for ATP sensing over other representative anions and discriminated it from adenosine 5?-diphosphate (ADP) and adenosine 5?- monophosphate (AMP). This can be explained by the much stronger electrostatic interactions between the polymer and ATP than the interactions between the polymer and ADP or AMP, as confirmed through molecular dynamics simulations.