The interplay between mitochondria and lipid droplets (LDs) plays a central role in regulating the β-oxidation and storage of fatty acids (FA) and is also engaged in responding to external stimuli such as nutrient deficiency. However, a single fluorescent probe enabling the discriminative and simultaneous visualization of the two organelles has not been reported yet, which brings limitation for the in-depth study on their interplay. In this work, utilizing the intramolecular spirocyclization reaction of rhodamine dyes that can dramatically change the optical and soluble properties, we have designed a new single fluorescent probe for labeling LDs and mitochondria in clearly separated dual-emission channels. The newly designed "biform" probe, , presented in a ring-opened form in mitochondria to give a strong red emission, while it underwent the intramolecular spirocyclization reaction to target LDs showing an intense blue fluorescence. In this manner, can label LDs and mitochondria in blue and red fluorescence, respectively. With this robust probe, the increase of mitochondria-LD contact and peridroplet mitochondria (PDM) amount during oleic acid treatment and starvation-induced autophagy has been successfully revealed. The interaction between the two organelles was also visualized in different tissues, which revealed an obviously higher level of mitochondria-LD contact and PDM amount in brown adipose tissue and lung tissue. This work provides a promising molecular tool to investigate the interplay between mitochondria and LDs and promotes studies on FA metabolism and autophagy.