A novel peptide fluorescent chemosensor (H2L) with a lysine backbone and both -NH2 sites conjugated with cysteine and dansyl groups has been designed and synthesized by solid phase peptide synthesis with Fmoc chemistry. This chemosensor is a promising analytical tool for detecting Cd(2+) based on the photo-induced electron transfer (PET) effect by turn-on response in 100% aqueous solutions. As designed, H2L exhibits excellent cell permeation and low biotoxicity as well as displaying relatively high selectivity and sensitivity. The chemosensor penetrated live HeLa cells and detected intracellular Cd(2+) by turn-on response. The binding stoichiometry and affinity, interference test, pH sensitivity, fluorescence quantum yield, quantum mechanical calculations, lifetimes, and cytotoxicity of the chemosensor H2L to Cd(2+) were also investigated. Moreover, H2L exhibits low biotoxicity with a limit of detection (LOD) for Cd(2+) of about 52 nM, implying that H2L can be used as a highly selective and sensitive peptide fluorescent chemosensor in biological systems.