A novel fluorescent bis-calix[4]arene macrocycle 9 incorporating metal-binding pockets was successfully prepared. The structure of macrocycle 9 and its precursors were characterized via EI-MS, MALDI-TOF-MS, ESI-MS, H-1 NMR, (CNMR)-C-13, 2D NMR, and X-ray crystallography. The macrocycle 9 displayed selective fluorescence quenching after interacting with Cu2+ in the presence competing metal cations including Mg2+, Ca2+, Ba2+, Ag+, Zn2+, Ti4+,Cd2+, Hg2+, Pb2+, In3+, La3+, Cr3+, Ni2+, Sb3+, V5+, Fe3+, Co2+, Sn2+, Sn2+, and Tl+. The Cu2+ limit of detection was found to be 40 nM much lower than its threshold level (similar to 20 mu M) in drinking water permitted by the U.S Environmental Protection Agency (EPA). Furthermore, drinkingwater samples from Karachi University (Pakistan) spiked with Cu2+ were analysed with the sensing system and the results showed an excellent agreement with the fluorescence quenching phenomenon of macrocycle 9 examined in deionized water. Importantly, the chemosensor 9 could be used to detect Cu2+ in living cells (C) 2016 Elsevier B.V. All rights reserved.