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[Display omitted] • A novel rhodamine B-based chemosensor has been developed for Cu(II) detection. • The chemosensor produces pronounced naked-eye and fluorescence responses. • The chemosensor has a high absolute fluorescence quantum yield of 77% • The chemosensor is selective for Cu(II) ions in complex matrices. • The chemosensor is suitable for fluorescence imaging in living cells. The regulation of copper in the human body is important for the prevention of several diseases. Therefore, a rhodamine B-based chemosensor 1 that demonstrates substantial affinity and selectivity was synthesized for the fluorescence imaging of copper(II). In the presence of Cu2+, the chemosensor underwent a color change from colorless to amaranth that was visible to the naked eye, and the fluorescence intensity did not change when excess EDTA was added to the solution. Furthermore, strong fluorescence was observed at 575 nm. The limit of detection was determined as 12.1 nM. The absolute fluorescence quantum yield was as high as 77 % and the stoichiometry between 1 and Cu2+ was determined to be 1:1 using a job plot. An analytical method was developed and successfully used to evaluate the sensor's ability for the fluorescence imaging of Cu2+ in HeLa tumor cells. [ABSTRACT FROM AUTHOR]