Here, sister calix[4]arene derivatives containing two NBD (7-nitro-benzofurazan) units in 1,3-(distal) derivatives (sensors 4&8) were prepared and characterized by 1H, 13C NMR, FTIR, elemental analysis, and fluorescence spectroscopy. The ion binding properties of multi-channel sensors 4&8 were performed in the presence of various cations and anions. Both sensor compounds, 4&8, showed “on–off” type fluorescence response towards Cu2+ cation and H2PO4−&F− anions with high selectivity in aqueous solution (CH3CN/H2O, 4/1, v/v). The connected spectroscopic parameters were investigated as the complex stoichiometry, binding constant etc. for a better result. Moreover, the lowest detection limit of 4 for sensing Cu2+ and H2PO4−&F− were obtained 2.4 × 10−8, 9.1 × 10−7, 9.7 × 10−7 mol/L respectively. the lowest detection limit of Cu2+ and H2PO4−&F− by 8 was obtained as 2.9 × 10−8, 9.7 × 10−7, 8.9 × 10−7 mol/L respectively. The confocal microscope was performed to investigate the sensor properties of 4&8 towards ions on living cells. Sensors 4 and 8 showed fluorescence properties on the A549 cell line, while it was observed to turn off the fluorescence intensity in cells treated with H2PO4−, F− and Cu2+ ions. Besides, compounds 4 and 8 can be used to identify H2PO4−, F− and Cu2+ ions in living cells.