Cooperation between organelles is essential to maintain the normal operation of the cell. A lipid droplet (LD), a dynamic organelle, is specialized in lipid storage and can interact physically with mitochondria in several cell types. However, an appropriate method for in situ studying the interaction relationships of mitochondria–LDs is still lacking. Herein, a charge-dependent strategy is proposed for the first time by considering adequately the charge difference between mitochondria and LDs. According to the novel strategy, we have developed a unique fluorescent probe Mito–LD based on the cyclization and ring-opening conversion. Mito–LD could simultaneously stain mitochondria and LDs and emit a red and green fluorescence, respectively. More importantly, with the probe Mito–LD, the in situ interaction relationships of mitochondria–LDs were investigated in detail from LD accumulation, mitochondrial dysfunction, lower environmental temperatures, and four aspects of apoptosis. The experimental results showed that mitochondria played an important role in LD accumulation, and the numbers and size of LDs would increase after mitochondrial dysfunction that may be due to excess liposomes. In addition, as an energy storage organelle, LDs played an important role in helping to coordinate mitochondrial energy supply in response to cold. In addition, the Mito–LD revealed that the polarity of mitochondria was higher than that of LDs. In a word, the probe Mito–LD could serve as a potential tool for further exploring mitochondria–LD interaction mechanisms, and importantly, the charge-dependent strategy is valuable for designing robust new probes in imaging multiple organelles.