Time-gated luminescence (TGL) bioassay technique using luminescent lanthanide complexes as probes has exhibited excellent practicability in detection of various analytes in autofluorescence-rich biosamples. Herein, a novel copper(II)-coupled lanthanide complex-based luminescence probe has been developed for the ratiometric TGL detection of hydrogen sulfide (H2S) in vitro and in vivo. The probe is constructed based on a dual-functional ligand that was synthesized by conjugating di(2-picolyl)amine with terpyridine polyacid (DATP) using the "click chemistry" method. The as-prepared ligand can coordinate to Eu3+ and Tb3+ ions with its terpyridine polyacid moiety to display long-lived emissions of Eu3+ and Tb3+ ions, while the further coordination of its di(2-picolyl)amino moiety to Cu2+ induces to the luminescence quenching of the Eu3+ complexes, which allows the probe consisting of the heterometallic Cu2+-DATP-Eu3+/Tb3+ complexes to be constructed. After the probe was reacted with H2S to lead to the release of Cu2+ from the complexes, the emission of Eu3+ at 610 nm was remarkably enhanced, while that of Tb3+ at 540 nm was changed slightly. This luminescence response feature allowed the probe to be conveniently applied for the ratiometric TGL determination of H2S with I610/I540 as the signal. The applicability of the probe for quantitative detection of H2S in human sera as well as for imaging of H2S in living cells and zebrafish were evaluated. All of the results proved the potential of the probe for in vitro and in vivo applications.