Tobacco mosaic virus (TMV) is a severe worldwide disease and capable of greatly reducing tobacco quality and yield. In this study, we developed the loop-mediated isothermal amplification (LAMP) method for the specific detection of this virus. Mg2+ concentrations, the reaction temperature, and the reaction time of LAMP were optimized to 6 mM, 65 °C, and 60 min, respectively. The detection limit of the LAMP method was as low as 7 copies and was 100 times more sensitive than the conventional PCR technique. Visual inspection of LAMP amplifications demonstrated that the positive and negative reactions exhibit distinct and different colours in daylight, which means that gel electrophoresis is not necessary to judge the presence or absence of the virus. LAMP can be conducted in 1 h and requires only a simple heating device for incubation. Thus, the LAMP-TMV detection protocol has great potential for use in the detection of TMV in both the laboratory and the field.