The invention relates to molecular detection technology of microsporidian on infected bumble bees, which belongs to the technical field of animal inspection and quarantine and particularly relates to the technical field of molecular inspection for accurately and quickly detecting the microsporidian on imported and exported bumble bees. The molecular detection technology is characterized in that aChelex-100 method is adopted to extract DNA; specific primers are used for carrying out PCR for one time; SSUrRNA genetic fragments of 540bp microsporidian are amplified; then, enzyme digestion of PCR products is carried out by restriction endonuclease Afa I; electrophoretic detection of enzyme digestion products are carried out by 2.0 percent agarose gel; and the type of the microsporidian parasitizing on the bumble bees can be detected directly through the length of fragments of the enzyme digestion products. The molecular detection technology can be applied to the inspection and quarantineof pathogen microsporidian on the imported and exported bumble bees.