Fluorescence-based single-vesicle fusion assays provide a powerful method for studying mechanisms underlying complex biological processes of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated vesicle fusion and neurotransmitter release. A crucial element of these assays is the ability of the fluorescent probe(s) to reliably detect key intermediate events of fusion pore opening and content release/mixing. Here, we report a new, reliable, and efficient single-vesicle content-mixing assay using a high affinity, fluorophore tagged; host-guest pair, cucurbit[7]-uril-Cy3 and adamantane-Cy5 as a fluorescence resonance energy transfer (FRET) pair. The power of these probes is demonstrated by the first successful observation of, flickering dynamics of the fusion pore by in vitro assay using neuronal SNARE-reconstituted vesicles.