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Water-soluble conjugated polymers (WCPs) composed of a hydrophobic polythiophene main chain with hydrophilic tertiary amine side-chains can directly self-assemble into sphere-like nano-objects in an aqueous solution due to phase separation between the hydrophilic and hydrophobic segments of the polymeric structure. Due to the presence of gas-responsive tertiary amine moieties in the spherical structure, the resulting polymers rapidly and reversibly tune their structural features, surface charge, and fluorescence performance in response to alternating carbon dioxide (CO2) and nitrogen (N2) bubbling, which leads to significantly enhanced fluorescence and surface charge switching properties and a stable cycle of on and off switching response. In vitro studies confirmed that the CO2-treated polymers exhibited extremely low cytotoxicity and enhanced cellular uptake ability in normal and tumor cells, and thus possess significantly improved fluorescence stability, distribution, and endocytic uptake efficiency within cellular organisms compared to the pristine polymer. More importantly, in vivo assays demonstrated that the CO2-treated polymers displayed excellent biocompatibility and high fluorescence enhancement in living zebrafish, whereas the fluorescence intensity and stability of zebrafish incubated with the pristine polymer decreased linearly over time. Thus, these CO2 and N2-responsive WCPs could potentially be applied as multifunctional fluorescent probes for in vivo biological imaging.