In the prodrug research field, information obtained from traditional end point biochemical assays in drug effect studies could provide neither the dynamic processes nor heterogeneous responses of individual cells. In situ imaging microscopy techniques, especially fluorescence lifetime imaging microscopy (FLIM), could fulfill these requirements. In this work, we used FLIM techniques to observe the entry and release of doxorubicin (Dox)-Cu complexes in live KYSE150 cells. The Dox-Cu complex has weaker fluorescence signals but similar lifetime values as compared to the raw Dox, whose fluorescence could be released by the addition of biothiol compound (such as glutathione). The cell viability results indicated that the Dox-Cu compound has a satisfactory killing effect on KYSE150 cells. The FLIM data showed that free doxorubicin was released from Dox-Cu complexes in cytoplasm of KYSE150 cells and then accumulated in the nucleus. After 90 min administration, the fluorescence lifetime signals reached 1.21 and 1.46 ns in the cytoplasm and nucleus, respectively, reflecting the transformation and transportation of Dox-Cu complexes. In conclusion, this work provides a satisfactory example for the research of prodrug monitored by FLIM techniques, expanding the useful applications of FLIM technique in drug development.