Migrasomes, recently identified as extracellular vesicles formed along the retraction fibers (RFs) in migrating cells, are evident in damaged mitochondria clearance, intercellular signaling, embryonic development, and pathological progression of diseases. During migrasome biogenesis, signaling cues and membrane dynamics interplay with each other in the maturation, expansion, and detachment of migrasomes from RFs. Membrane tension plays a crucial role in migrasome dynamics, and tools for monitoring the dynamic mechanical properties of migrasomes are highly desired. In this study, we developed and characterized a novel mechanosensitive but viscosity-insensitive fluorescent probe, named OT2SQ, for real-time investigation of migrasome dynamics in living cells. OT2SQ is a twisted small molecule with multiple rotatable units, exhibiting longer fluorescence lifetimes in response to increased membrane tension but showing no response to altered viscosity. Using time-lapse fluorescence lifetime imaging, we identified distinct modes of migrasome biogenesis and detachment from retraction fibers, each associated with unique membrane tension dynamics. Furthermore, phasor-FLIM analysis revealed alterations in migrasome membrane tension during content release and internalization processes. This study introduces a versatile fluorescent tool for real-time monitoring of migrasome morphology, spatiotemporal distribution, and membrane tension dynamics.