Lead is one of the most toxic substances. However, there are few ratiometric fluorescent probes for sensing Pb²⁺ in aqueous solution as well as living cells because specific ligands for Pb²⁺ ions have not been well characterized. Considering the interactions between Pb²⁺ and peptides, we developed ratiometric fluorescent probes for Pb²⁺ based on the peptide receptor in two steps. First, we synthesized fluorescent probes (<b>1</b>-<b>3</b>) based on the tetrapeptide receptor (ECEE-NH₂) containing hard and soft ligands by conjugation with diverse fluorophores that showed excimer emission when they aggregated. After investigation of fluorescent responses to metal ions, benzothiazolyl-cyanovinylene was evaluated as an appropriate fluorophore for ratiometric detection of Pb²⁺. Next, we modified the peptide receptor to decrease the number of hard ligands and/or to replace Cys with disulfide bond and methylated Cys for improving selectivity and cell permeability. From this process, we developed two fluorescent probes (<b>3</b> and <b>8</b>) among the probes (<b>1</b>-<b>8</b>) that exhibited remarkable ratiometric sensing properties for Pb²⁺ including high water solubility (≤2% DMF), visible light excitation, high sensitivity, selectivity for Pb²⁺, low detection limits (<10 nM), and fast response (<6 min). The binding mode study revealed that specific Pb²⁺-peptide interactions of the probes caused nanosized aggregates in which the fluorophores of the probes came close each other, exhibiting excimer emission. In particular, <b>8</b> based on tetrapeptide bearing a disulfide bond and two carboxyl groups with a good permeability successfully quantified intracellular uptake of Pb²⁺ in live cells through ratiometric fluorescent signals. The ratiometric sensing system based on specific metal-peptide interactions and excimer emission process could provide a valuable tool to quantify Pb²⁺ in live cells and pure aqueous solutions.