In this paper, the interaction of the antibacterial peptide MDL-1 of Musca domestica L and E. coli DNA was investigated by fluorescence spectra. The interaction mode was studied by using ethidium bromide (EB) as an extrinsic fluorescence probe. The result of fluorescence spectra and Scatchard plot indicate that the binding constant and the number of binding sites between DNA and EB decrease with increasing concentration of MDL-1. The change in binding constant and binding sites showed that the conformation of DNA transformed. When the EB molecule just fitted into the interval, the fluorescence intensity was maximum. When MDL-1 was present in the system, the EB molecules was excluded from DNA, then the fluorescence intensity decreased. The result showed that the style was eletrostatic binding, groove binding and intercalation in the interaction of MDL-1 and double helix DNA. The binding constant of MDL-1 complex with DNA was determined. Meanwhile, it helped to explain the molecule mechanism of antibacterial peptides from the interaction style and structure characteristic of antibacterial peptide and bacterial DNA.