A cationic water-soluble conjugated polyelectrolyte, poly[9,9-bis(6''-(N,N,N-trimethylammonium)hexyl)fluorene-co-alt-2,5-bis(6'-(N,N,N-trimethylammonium)hexyloxyphenylene) tetrabromide], was synthesized. Fluorescence resonant energy transfer (FRET) experiments between the polymer and fluorescein-labeled single-stranded DNA (ssDNA-Fl) were conducted in aqueous buffer and THF/buffer mixtures. Weak fluorescence emission in aqueous buffer was observed upon excitation of the polymer, whereas addition of THF turned on the fluorescence. Fluorescence self-quenching of ssDNA-Fl in the ssDNA-Fl/polymer complexes as well as electron transfer from the polymer to fluorescein may account for the low fluorescence emission in buffer. The improved sensitization of fluorescence by the polymer observed in THF/buffer could be attributed to the weaker binding between the polymer and ssDNA-Fl and a decrease in dielectric constant of the solvent mixture, which disfavors electron transfer. THF-assisted signal sensitization was also observed for the polymer and fluorescein-labeled double-stranded DNA (dsDNA-Fl). These results indicate that the use of cosolvent provides a strategy to improve the detection sensitivity for biosensors based on the optical amplification provided by conjugated polymers.