Organelles are dynamic yet highly organized to preserve cellular homeostasis. However, the absence of time-resolved molecular tools for simultaneous dual-signal imaging of two organelles has prevented scientists from elucidating organelle interaction regulatory mechanisms on a nanosecond timescale. To date, the regulatory mechanisms governing the interaction between endoplasmic reticulum (ER) and autophagosomes are unknown. In this study, we propose a strategy for developing dual-fluorescence lifetime probes localized to the endoplasmic reticulum and autophagosomes to investigate their interaction regulatory mechanisms. Using the robust probe CF2, we investigated the regulatory mechanisms between ER and autophagosomes and discovered the following: (i) motile autophagosome in ER tips drives the ER tubule to grow and slide; (ii) the ER reticulate tubule forms a three-way junction centered on the autophagosome; (iii) ER autophagy is a type of cell damage index during drug-induced apoptosis. Thus, this study advances our knowledge of organelle interaction regulatory mechanisms, shedding light on the identification of therapeutic targets for neurodegenerative diseases.