In this work, the first CdSe/ZnS quantum dot (QD) photoluminescence lifetime based chloride ion nanosensor is reported. The acridinium dication lucigenin was self-assembled on the surface of negatively charged mercaptopropionic acid capped QDs to achieve QD–lucigenin conjugates. Upon attachment, a drastic decrease of the photoluminescence lifetime of both QD nanoparticles and lucigenin is observed by virtue of a charge transfer mechanism. Since lucigenin is a chloride-sensitive indicator dye, the photoluminescence decay of QD–lucigenin conjugates changes by adding chloride ion. The photoluminescence lifetime of the QDs in the conjugate increases after reacting with Cl⁻, but also shows a concomitant decrease in the lucigenin lifetime immobilized on the surface. The photoluminescence lifetime of QD–lucigenin nanosensors shows a linear response in the Cl⁻ concentration range between 0.5 and 50 mM. Moreover, the ratio τ_ave^QD/τ_ave^luc can be used as an analytical signal since the lifetime ratio presents a linear response in the same Cl⁻ concentration range. The system also shows good selectivity towards most of the main anions and molecules that can be found in biological fluids. These nanosensors have been satisfactorily applied for Cl⁻ determination in simulated intracellular media with high sensitivity and high selectivity. Finally, we demonstrate the potential application of the proposed nanosensor in confocal fluorescence lifetime imaging (FLIM). These results show the promising application of the QD–lucigenin nanosensors in FLIM, particularly for intracellular sensing, with the invaluable advantages of the time-resolved fluorescence techniques.