Hydrogen sulfide (H2S) has been recognized as an important endogenous gasotransmitter associated with biological signaling transduction. However, recent biological studies implied that the H2S-related cellular signaling might actually be mediated by hydrogen polysulfides (H2S n , n > 1), not H2S itself. Unraveling such a mystery strongly demanded the quantification of endogenous H2S n in living systems. However, endogenous H2S n has been undetectable thus far, due to its extremely low concentration within cells. Herein, we demonstrated a strategy to detect ultra-trace endogenous H2S nvia a fluorescent τ-probe, through changes of fluorescence lifetime instead of fluorescence intensity. This τ-probe exhibited an ultrasensitive response to H2S n , bringing about the lowest value of the detection limit (2 nM) and a lower limit of quantification (10 nM) to date. With such merits, we quantified and mapped endogenous H2S n within cells and zebrafish. The quantitative information about endogenous H2S n in cells and in vivo may have a significant implication for future research on the role of H2S n in biology. The methodology of the τ-probe established here might provide a general insight into the design and application of any fluorescent probes, beyond the limit of utilizing fluorescence intensity.