In this work, a dumbbell-shaped bi-acetylferrocene ethylenediamine complex, (AFc)(2)-en (AFc = acetylferrocene, en = ethylenediamine) was synthesized and its interaction with double-stranded DNA (dsDNA) was comprehensively studied by electrochemical methods on a gold electrode. The experimental results reveal that the highly electroactive (AFc)(2)-en complex can interact with dsDNA via a specific bis-groove binding mode. The binding constant and binding site size between the two species were determined to be 3.7 x 10(5) L mol(-1) and 4.2, respectively. The surface-based studies further show that (AFc)(2)-en presents the stronger electrochemical response on dsDNA modified electrode than on the single-stranded DNA (ssDNA) modified electrode, suggesting that the complex can well distinguish dsDNA from ssDNA. Based on this characteristic, the bi-acetylferrocene complex was utilized as an electrochemical hybridization indicator in a DNA biosensor for the detection of oligonucleotides from CaMV35S promoter gene. The specificity experiments indicate that the biosensor can well discriminate the complementary sequence from the base-mismatched and the non-complementary sequences. The complementary target sequence can be quantified over a wide range from 1.0 x 10(-14) mol L-1 to 1.0 x 10(-6) mol L-1 with a detection limit of 2.0 x 10(-15) mol L-1. (c) 2013 Elsevier Ltd. All rights reserved.