An impedimetric DNA biosensor based on nanofiber mats were fabricated for the detection of Salmonella spp. Polyurethane/Poly(m-anthranilic acid) (PU/P3ANA) nanofibers were obtained by electrospinning. Capture DNA probe was covalently immobilized onto PU/P3ANA nanofibers by activation of carboxyl groups on the nanofibers. The hybridization was carried out with fully complementary and mutant target DNA with a single or double-base mismatch. The immobilization and hybridization process was investigated by electrochemical impedance spectroscopy (EIS) and the data were fitted to equivalent electrical circuit model (ECM). The hybridization of complementary DNA with the immobilized probe DNA increased the charge transfer resistance of the PU/P3ANA nanofibers. The fabricated DNA biosensor was stable up to one month and displayed linear response (0.1 mu M - 10 mu M), high selectivity (8.17 k Omega/mu M) and single-base mismatch selectivity. This study represents a sensitive and advanced nanofiber based DNA biosensor for the detection of Salmonella spp. by EIS. (C) 2017 Elsevier B.V. All rights reserved.