The present work deals with interplay of proflavine (PF) in between the nano-cavity of a supramolecular host, cucurbit[7]uril (CB7) and DNA by monitoring the fluorescence changes of proflavine using steady-state and picosecond time-resolved spectroscopic techniques. Upon encapsulation in CB7 nano-cavity, PF shows a prominent enhancement in emission maximum with a substantial blue-shift, whereas introduction of DNA into the same system (PF+CB7) brings very negligible change in the emission profile. These results indicate that the PF molecule cannot be released to DNA without introducing external stimulus. However, with addition of acetylcholine, the fluorescence intensity decreases rapidly with a significant red-shift. And these spectral features exactly match with emission spectrum of PF with only DNA. Time resolved fluorescence and anisotropy results further confirm that an external stimuli like acetylcholine is necessary for this translocation of the guest from CB7 to DNA. This kind of simple supramolecular controlled release of the PF may find suitable applications in future for designing new CBn based drug carriers. © 2015 Elsevier B.V. All rights reserved.