Two new hydroxynaphthyl-hydrazone based fluorogenic chemosensors R¹ and R² have been synthesized by Schiff base condensation of Tris(4-formylphenyl)amine with 1-hydroxynaphthalene-2-hydrazide and 1-hydroxynaphthalene-2-carbohydrazone, respectively. They are examined as highly selective and sensitive receptors for Cu²⁺ ions in aqueous medium. Electronic absorption as well as fluorescence titration studies of receptors R¹ and R² with different metal cations in H₂O/CH₃CN medium showed highly selective and very rapid (<2 min) binding affinity towards Cu²⁺ ions even in the presence of other commonly coexisting metal ions such as Na⁺, K⁺, Mg²⁺, Ca²⁺, Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺ and Hg²⁺. Quantification of the fluorescence titration analysis indicated that these newly synthesized receptors (R¹ and R²) can indicate the presence of Cu²⁺ ions even at very low concentrations of 598 and 676 ppt, respectively. In addition, the propensity of these receptors as bio-imaging fluorescent probes to detect Cu²⁺ ions in human cervical HeLa cancer cell lines and their cytotoxicity against HeLa cells have been investigated.