High interest in the detection and quantification of (elevated) intracellular zinc levels in tumor cells has been aroused due to the crucial physiological roles and the pathophysiological implications of zinc. Despite their diverse applications, benzo[1,2-b:4,5-b′]difuran (BDF) derivatives as a fluorescent Zn(II) probe in living cells have not been reported. By careful chemical design of molecular components, we prepared and fully characterized a new amphiphilic BDF-based fluorophore. A series of experiments were carried out to test the cytotoxicity and cellular uptake behavior in human cancer derived HeLa cells. The ligand fluorescence is strongly quenched upon the addition of Zn(II) ions. The high cell membrane permeability, low cytotoxicity and significant fluorescence quenching behavior render this ligand very promising for in vitro detection of Zn(II) ions in biological systems.