We report the design and synthesis of a new perylene bisimide derivative containing calix[4]arene units (PB-CX[4]) as "turn on" fluorescent sensor for Hg2+ ion determination. PB-CX[4] showed highly selective and sensitive "turn-on" fluorescent responses toward Hg2+ ion based on photoinduced electron transfer (PET) mechanism in DMF/H2O (v/v, 95/5). The binding analysis using a Job plot suggested that PB-CX[4] formed a 1:2 complex with Hg2+. The association constant (K) of PB-CX[4]-Hg2+ complex was found to be 1.66 × 109 M-2, with a detection limit of 5.56 × 10-7 M. In addition, possible utilization of PB-CX[4] as bio-imaging fluorescent probe to detect Hg2+ in human colon cancer cell lines was also observed by confocal fluorescence microscopy.