Three novel and facile calixarene derivatives (5, 6 and 7), which were appended with four rhodamine units at the upper rim of calixarene skeleton, were firstly prepared and evaluated for selective detection of metal ions in solution. Receptors (5) and (7) indicated immediate turn on fluorescence output toward Hg2+ ions over other most competitive metal ions with the ultralow detection limits, indicating their high efficiency and reliability. The binding response to Hg2+ ions in solution was also observed through a chromogenic change (from colorless to pale pink). Furthermore, in vitro and bio-imaging studies with MCF-7 or MIA PaCa-2 cell lines were also performed to investigate the use of receptors in biological systems in order to monitor of mercury ions. Results showed that new receptors (5) or (7) were cell permeable and suitable for real-time imaging of Hg2+ in living cells (MCF-7) or (MIA PaCa-2) without any damage to healthy cell lines (HEK 293).