A new cationic cyano-substituted poly(p-phenylenevinylene) (N-CNPPV) is synthesized by Knoevenagel condensation. The water-soluble polymer shows different emission spectra in different solvents and displays unique fluorescent behaviors in the mixed solvents of water and THF. The new polymer can form a complex with ssDNA by adopting a more planar conformation, exhibiting red shift of emission wavelength and enhancement of fluorescence intensity. By investigating the fluorescent response of N-CNPPV to various surfactants, we demonstrate that the hydrophobic interaction and electrostatic interaction result in the selective response of N-CNPPV to ssDNA. This is the first report on selective fluorescence enhancement of conjugated polyelectrolyte induced by ssDNA.