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[Display omitted] • A new fluorescent probe DAHK was synthesized based on copper peptide backbone. • DAHK could detect Cu2+ in 100 % aqueous medium with obvious fluorescence quenching. • DAHK-Cu2+ ensemble exhibited fluorescent "off–on" response with H 2 S. • DAHK was successfully used to selectively detect Cu2+ and H 2 S in living cells. • DAHK was successfully applied to sensitively detect Cu2+ and H 2 S in test strips. Copper ions (Cu2+) and hydrogen sulfide (H 2 S) detection is of great significance in environmental monitoring and disease screening. Herein, a novel fluorescent probe (DAHK) was rationally designed and easily synthesized based on a copper peptide backbone labelled a dansyl fluorophore. DAHK exhibited large Stokes shift (210 nm), high sensitivity (38.9 nM) and high selectivity response to Cu2+ ions based on obvious fluorescence quenching in HEPES buffer (10.0 mM, pH 7.4) solutions. In addition, the in situ formed DAHK -Cu2+ ensemble was successfully used as a secondary probe for rapidly and sequentially detected H 2 S via fluorescence "off–on" response, and without interference by a range of other anions. The limit of detection (LOD) for 47.3 nM was determined, which was a much lower value than WHO and EPA guidelines (H 2 S) for drinking water. The reversibility of interaction of DAHK with Cu2+ and H 2 S was monitored using fluorescence emission spectra. Cells imaging experiments exhibited that DAHK had good membrane permeability and excellent low toxicity. Moreover, DAHK was successfully made into fluorescent test strips for visual detection and rapid analysis based on the exciting stability and excellent water solubility. [ABSTRACT FROM AUTHOR]