Stonebrood and Chalkbrood are major fungal diseases in honeybee (Apis mellifera), caused by Aspergillus flavus, or by Ascosphaera apis, respectively. Because of similar shapes and symptoms of infected larvae by them, it could be hard to distinguish between both without gene analysis. In this study, we developed a Loop-mediated isothermal amplification (LAMP) method for the specific detection of A. flavus in honeybee. Optimal conditions for A. flavus-specific LAMP were sequentially established using the specific primer sets, RT-A. flavus-F3/ B3/ FIP1 /BIP2. The existence of A. flavus among genomic DNA from honeybee was detected by A. flavus-specific LAMP within 27 minutes, and the detection limit was estimated 105 DNA molecules of target DNA. Specific amplification of A. flavus-specific LAMP can be recognized with colorimetric dyes by naked eyes. It makes easy screening and time-saving to identify A. flavus-infection in field tests. This assay would be useful not only for A. flavus detection in honeybee in laboratory, but also could be applied in apiary field.