Real-time monitoring of dynamic microvesicles (MVs), vesicles associated with living cells, is of great significance in deeply understanding their origin, transport, and function. However, specific labeling MVs poses a challenge due to the lack of unique biomarkers that differentiate them from other cellular compartments. Here, we present a strategy to selectively label MVs by evaluating a series of lipid layer-sensitive cationic indolium-coumarin fluorescent probes (designated as IC-Cn, with n ranging from 1 to 18) that feature varying aliphatic side chains (CnH2n+1). Through in situ cell imaging and analysis, we found that IC-Cn location is highly related to their lipophilicities and the phospholipid layer hydrophobic microenvironments in cellular compartments. In detail, IC-C1 and IC-C2 specifically localize MVs both inside and outside cells. In contrast, IC-C3, IC-C4, and IC-C5 label cellular MVs and mitochondria but with distinct fluorescence lifetimes. Using these probes strategically, we have discovered that, in addition to the biogenesis of MVs from plasma membranes and damaged mitochondria, newly formed MVs can undergo fusion and fission processes. Moreover, mitochondria-derived MVs, beyond being released from parent cells, can fuse with lysosomes to facilitate the removal of dysfunctional mitochondria. The work not only provides new insights into MV physiology but also inspires the design strategies for probes used in specific labeling in cell studies.