In this study, a novel colorimetric and fluorescent dual-functional peptide-based probe FAHF was rationally designed and successfully prepared using solid phase peptide synthesis (SPPS) technology. As designed, FAHF exhibited high selectivity and rapid response towards Cu²⁺ and Hg²⁺ with colorimetric and fluorescent dual-signal sensing mode in 100% aqueous media, and the recognizable color change was clearly observed with naked eye under daylight lamp and 365 nm UV light. In addition, the low concentrations of Cu²⁺ and Hg²⁺ could be accurately detected with the limit of detection (LODs) for 61.02 nM and 40.27 nM, respectively. At the same time, due to the strong coordination of Cu²⁺/Hg²⁺ with S²⁻, the FAHF-Cu²⁺ ensemble and FAHF-Hg²⁺ ensemble formed in situ were successfully used as two new promising cascade probes for rapidly and sequentially detecting S²⁻ based on colorimetric method and fluorescence “off-on” response, and the corresponding limit of detection (LODs) were calculated to be 45.12 nM and 102.38 nM, respectively. Moreover, FAHF was successfully applied for fluorescence imaging of Cu²⁺, Hg²⁺ and S²⁻ in two living cells due to good cell permeability and low cytotoxicity. Finally, FAHF was successfully made into test strips for visual detection and rapid analysis under 365 nm UV lamp, and smartphone-assisted visual assays were successfully used to evaluate and monitor the Cu²⁺ levels with a limit of detection (LOD) for 0.62 μM.