A novel fluorescent chemosensor with dansyl-appended tetrapeptide (identified as L), was designed and synthesized using solid phase synthesis (SPPS) technology. At room temperature, L displayed high water solubility and highly selective fluorescence âturn-onâ response towards Zn2+ detection in 100% aqueous solutions. Conversely, L exhibited highly selective detection of Cu2+ through fluorescence quenching by a mechanism intrinsic to the paramagnetic species. The binding stoichiometry with L and Zn2+/Cu2+ ions were determined from the Jobâs plot to be 2:1, and further verified with fluorescence titration, ESI-HRMS and DFT-Calculations studies. The lowest measured detection limits for L were 13.2 nM for Zn2+ and 4.1 nM for Cu2+, respectively, with excellent sensitivity. In addition to that, L was successfully analyzed quantitatively for Zn2+ and Cu2+ ions using logic gate construction. Furthermore, L exhibited excellent cell permeation and low biotoxicity during bioimaging studies, L was able to selectively identify Zn2+ and Cu2+ ions in living LNcap cells, suggesting that the chemosensor L will be a promising biomolecular chemosensor for multifunctional detection of Zn2+ and Cu2+ through differential responses in environmental and biological systems.