The invention discloses a tobacco bacterial wilt real-time fluorescence quantitative PCR detection kit and detection method, and belongs to the technical field of molecular biology. A bacterial wilt extracellular glucanase gene (EG) regional segment is used as a target detection sequence, a primer sequence covering the target segment is synthesized by adopting an LNA technology, and a fluorescence quantitative nucleic acid amplification detection kit which can high sensitively, efficiently and rapidly detect tobacco bacterial wilt by combining a fluorescence quantitative nucleic acid amplification technology. Compared with the ordinary method for detecting the tobacco bacterial wilt by virtue of primer conventional fluorescence quantitative PCR, the tobacco bacterial wilt real-time fluorescence quantitative PCR detection kit and detection method sensitized by locked-nucleic acid (LNA) are suitable for the quantitative detection of tobacco planting soil wilt bacteria and have advantages of high flux, high sensitivity, short detection time, simple operation process, relative low requirement on a sample to be detected, and the like.