The rhodamine based receptors were developed as colorimetric and fluorescent chemosensor towards Cu²⁺. By using UV–Vis and fluorescence spectroscopy techniques, their selectivity and photophysical properties towards various metal ions were determined. The obvious color change from colorless to pink (under normal light) and non‐fluorescent to yellowish‐green fluorescent (under UV light) were observed upon the copper addition. The metal binding structures were proposed based on analysis data of proton NMR and Job's plot. Cell imaging experiments were conducted to determine if the chemosensors could be used in biological systems. The results showed that cell viability did not decrease below 80% at any concentrations. Chemosensors and Cu²⁺ were added to the cells for cell imaging and fluorescence images. Overall, it was found that the chemosensors were cell‐permeable, non‐toxic, and functional in the cells.