Anthracnose caused by Colletotrichum gloeosporioides is an economic disease that affects soybean production worldwide. This study developed a rapid, sensitive method for the detection of C. gloeosporioides using a loop-mediated isothermal amplification (LAMP) assay. By targeting a glutamine synthetase (GS) gene sequence, the GS-Cg-LAMP assay works most efficiently at 64°C and allows the detection of C. gloeosporioides DNA within 70 min based on a color change from orange to yellow-green following the addition of SYBR Green I to the LAMP reaction products. The detection limit was 1 pg μL−1 of genomic DNA per reaction. In specificity tests, the positive reaction (yellow-green color by the naked eye) was observed only in the presence of C. gloeosporioides, and none of other Colletotrichum spp. or fungal isolates produced a color change. Moreover, the GS-Cg-LAMP assay was successfully used to diagnose anthracnose caused by C. gloeosporioides in diseased soybean samples obtained from the field and detected the pathogen in soybean seeds bought from farmers’ markets. Our study provides a simple, sensitive tool for the rapid diagnosis of soybean anthracnose caused by C. gloeosporioides.