The invention relates to fluorescent dye, a preparation method of the fluorescent dye and application of the fluorescent dye in bacterial detection. The fluorescent dye is combined with light reactivedye of dsDNA with high affinity, the fluorescence of the fluorescent dye is very weak, after being combined with nucleic acid, a fluorescent signal is very strong, cell membranes are impervious for the dye, the dye only can enter damaged cell membranes and can be boned with DNA through covalent bonds, and thus permanent DNA modification is caused. These modified DNAs can not be re-amplified, andtherefore, the method is combined with a PCR method in use can well make up for shortcomings of the a conventional detection method.