The present article deals with the detailed complexation study along with exploration of possible utilization of 25,26,27,28-tetra-hydroxy-11,23-di-(tert-butyl)-5,17-di-(2-antracenyl)azocalix[4]arene(7) and 25,26,27,28-tetra-hydroxy-11,23-di-(tert-butyl)-5,17-di-(1-pyreneyl)azocalix[4]arene (8) as bio-imaging fluorescent probe to detect Cu2+ in human colon cell line, SW-620, observed by fluorescence microscopy. Complexation properties of ligands 7 and 8 toward selected transition metal ions have been investigated by UV-vis and fluorescence spectroscopies. It has been noticed that ligands 7 and 8 are not only proved to be an efficient Cu2+selective chromoionophore but also possess effective fluorescence properties by complexing with Cu2+ ions. The FTIR spectroscopic method has also been applied for further confirmation of the complexation phenomenon of ligands 7 and 8 with metal ion and were found to be adequate. Moreover, fluorescence microscopy studies showed that ligand 7 may be used as fluorescent probe to detect Cu2+ in living cells.