In this work, a novel electrochemical DNA biosensor has been developed based on the hybrid film of fern leaf-like α-Fe 2 O 3 microparticles and chitosan (CS). The fern leaf-like α-Fe 2 O 3 microparticles were synthesized via a facile template-free hydrothermal method, and their morphologies were characterized by X-ray diffraction, energy dispersive spectrometry, scanning electron microscope, and transmission electron microscope. Electrochemical characterization assays revealed that the hybrid film modified electrode had remarkable synergistic effects of the large accessible surface area and high electrical conductivity of semiconductive Fe 2 O 3 , and the good film stability of CS. Based on the rich amino groups on CS, the CS–Fe 2 O 3 hybrid film was employed as a functional matrix for probe DNA immobilization using terephthalaldehyde (TPA) as a bifunctional arm-linker. The hybridization capacity of the developed biosensor was evaluated with electrochemical impedance spectroscopy (EIS) using [Fe(CN) 6 ] 3−/4− as the indicating probe. A wide dynamic detection range from 1.0×10 −14 to 1.0×10 −10 M with ultralow detection limit of 5.6×10 −15 M was achieved for the target DNA. The hybridization selectivity experiments further revealed that the biosensor could discriminate fully complementary sequences from one-base mismatched, three-base mismatched, and non-complementary sequences. Moreover, the biosensor showed the advantage of good regeneration ability and reproducibility.