A new conjugated poly(pyridinium salt) derivative (P1) containing heterocyclic phenanthridine moieties in the main chain with tosylate counterions was prepared via a ring-transmutation polymerization reaction. Its chemical structure was established using NMR and FTIR spectra. Its number-average molecular weight (Mn) and polydispersity were 14 430 and 1.456, respectively, as determined by gel permeation chromatography (GPC). The interaction of P1 with calf thymus DNA (ctDNA) in an ethanol-Tris-HCl buffer (2 mm, pH 7.4) (v/v=1:1) was investigated by ultraviolet–visible, fluorescence, circular dichroism spectroscopy, and dynamic light scattering (DLS) measurements. The electrostatic attraction and intercalation interaction between P1 and ctDNA resulted in a significant fluorescence quenching of polymer P1 in the ethanol–Tris-HCl buffer, and the ratio of fluorescence intensity at 523 nm in the absence of ctDNA (F0) to that in the presence of ctDNA (Fi) was linearly proportional to the concentration of ctDNA within the range of 0–6.0 μm. Therefore, the conjugated polyelectrolyte is expected to be used as a new fluorescent probe for the label-free detection of DNA. A new conjugated poly(pyridinium salt)s derivative (P1) containing heterocyclic phenathridine moieties in the main chain was prepared by ring-transmutation polymerization reaction. Its interaction with calf thymus DNA (ctDNA) in aqueous ethanol solution was explored by means of UV-vis, fluorescence, circular dichroism (CD) spectroscopy, as well as dynamic light scattering (DLS) measurements.