Milk vetch dwarf virus (MDV) is a member of the genus Nanovirus, and its genome is composed of multiple circular 1-kb ssDNA components. In this study, we first determined that the diseased tobacco samples obtained in Zhucheng, Shandong Province were naturally infected with MDV using a polymerase chain reaction (PCR) assay. Subsequently, loop-mediated isothermal amplification (LAMP) was developed for the detection of MDV for the first time. The Mg2+ and dNTP concentrations and the reaction temperature and time of the LAMP were optimized to 8 mM, 1.8 mM, 65 °C, and 60 min, respectively. The best ratio of the inner primers (FIP and BIP) to the outer primers (F3 and B3) was 2:1. The LAMP detection limit was 100 times greater than that of PCR. The nucleotide amplification could be clearly observed by adding SYBR Green I. The positive and negative reactions exhibit distinctly different colors in daylight; however, the positive reactions exhibit green fluorescence under a UV lamp. Therefore, the method is stable, sensitive and specific.