Polydiacetylene (PDA) has been extensively studied as "smart" sensing material. The bio-interaction induced chromism has been demonstrated for several bioanalytes. However the fluorescence-based signal transduction on PDA has not been widely explored for sensing application due to low quantum yield of the material. We reported a fluorescent sensor platform based on PDA vesicles and incorporated BODIPY lipid fluorophores, which has a fluorescence “on-off” switching property in response to external perturbations such as solution pH. The work was extended to a “mix-and-detect” type of fluorescent sensor for direct detection of bacterial cholera toxin (CT) utilizing the lipid insertion and the FRET mechanism. The strategy was also used for influenza virus detection. Synthetic influenza inhibitor, BCX, was attached to BODIPY lipid dye. BODIPY conjugated BCX (BO-BCX), dissolved in Tris buffer, demonstrated substantial fluorescence quenching with addition of PDA vesicle solution. The close proximity of the dye molecules to the conjugated chains as a result of lipid insertion enables energy transfer from dye to the polymer backbone, yielding the observed phenomenon. When influenza virus is present, the binding of BO-BCX to virus results in the formation of a complex that prohibits it from membrane insertion, leading to the blocking of the quenching process. The method is very simple and allows specific and sensitive detection of influenza virus with just a few mixing steps. It can be further developed into a general sensing strategy for detection of other analytes with amplified FRET mechanism.