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A biocompatible fluorogenic chemosensor (N -(3-carboxy acryloyl)) rhodamine B (RHA) comprising a furan-2,5-dione receptor and a rhodamine fluorophore via an iminohydrazine crosslinker has been prepared for more scrutinizing optical properties and utilization in molecular imaging of living cells. The consequences indicated that RHA not only is a good pH indicator in acidic media but also can be used as a great sensitive fluoroionophore with high selectivity. The fluorescence emission of the molecule RHA is quite stable in the presence of 15 diverse cations, 18 anions (especially H 2 PO 4 −, S2, and F−), and 5 amino acids with the different functional group as a model, except Cu2+ ions with observable emission intensity changing. The Cu2+ probe has a dual functional effect, an "on-off" mechanism in solvent media, and an "off-on" mechanism in Phosphate Buffered Saline (PBS) in acidic pH conditions. The detection limits for Cu2+ in acetone-water media was 0.69 μM and 0.18 μM in pH = 5.0. Moreover, RHA can fluorescently and colorimetrically image Cu2+ ions in acidic and neutral pH with low cytotoxicity in living cells. Unlabelled Image • The rhodamine-based chemosensor (RHA) has distinct fluorescent properties. • The fluorophore selectively targeted at Cu2+ ions among 15 different cations, 18 anions (specially F-, S2- and H 2 PO 4 -), and 5 amino acids. • The unique fluoroionophore probe has a dual property in Cu2+ fluorescence responsiveness, which immediately responded to Cu2+ ions in natural pH based on the CHEQ mechanism and acidic pH through fluorescence enhancement. • This probe exhibits excellent membrane-permeable, low cytotoxicity, and good performance in the detection of Cu2+ in the MCF7 cell line. [ABSTRACT FROM AUTHOR]