Here, a doxorubicin (Dox)-Cu complex is prepared and used as thiols-cleavable Dox prodrug in FLIM imaging studies. The Dox-Cu complex has no fluorescence and its absorption spectrum shows a significant red shift as compared to the raw Dox. When the complex is mixed with biothiol compound, the fluorescence of Dox is restored. In this paper, both Dox and Dox-Cu complexes are used in the fluorescence lifetime analysis, cell incubation and FLIM imaging studies. The way of intracellular transport and the changes in fluorescence lifetime of two compounds are basically the same. This indicates that the Dox-Cu complex has been decomposed into free Dox molecules and Cu-biothiol complex inside the cell. This research provides a valuable example for the study of Dox prodrug by FLIM technique, promoting the application of FLIM technique in drug research.