Research about DNA composition has been concentrated on DNA damage in the past few decades. However, it still remains a great challenge to construct a rapid, facile, and accurate approach for simultaneously monitoring four DNA bases, guanine (G), adenine (A), thymine (T), and cytosine (C). Herein, a novel electrochemical sensor based on phenanthroimidazole derivative, 2-(4-bromophenyl)-1-phenyl-1H-phenanthro[9,10-d]-imidazole (PPI), is successfully fabricated by a simple electrochemical method. The bromophenyl group in PI could expand their aromatic plane, induce the πconjugated extension, and enhance the charge transfer and π−π interaction. The phenyl group at N1 position could regulate the intermolecular interaction, which could promote the possibility of intermolecular connection. The PPI polymer (poly(PPI)) with π-electron enriched conjugation architecture has been applied in simultaneous determination of G, A, T, and C in neutral solution by square wave voltammetry (SWV) method with well-separated peak potentials at 0.714, 1.004, 1.177, and 1.353 V, respectively. The sensor functionalized with poly(PPI) exhibits wide linear response for G, A, T, and C in the concentration ranges of 3−300, 1−300, 30−800, and 20−750 μM, respectively. With favorable selectivity, stability, and reproducibility, the sensor is successfully utilized to monitor four DNA bases in real samples, displaying a promising prospect for electrochemical sensing devices.