Although multivalent glycosidase inhibitors have shown enhanced glycosidase inhibition activities, further applications and research directions need to be developed in the future. In this paper, two positional isomeric perylene bisimide derivatives (<b>PBI-4DNJ-1</b> and <b>PBI-4DNJ-2</b>) with 1-deoxynojirimycin conjugated were synthesized. Furthermore, <b>PBI-4DNJ-1</b> and <b>PBI-4DNJ-2</b> showed positional isomeric effects on the optical properties, self-assembly behaviors, glycosidase inhibition activities, and hypoglycemic effects. Importantly, <b>PBI-4DNJ-1</b> exhibited potent hypoglycemic effects in mice with 41.33 ± 2.84 and 37.45 ± 3.94% decreases in blood glucose at 15 and 30 min, respectively. The molecular docking results showed that the active fragment of <b>PBI-4DNJ-1</b> has the highest binding energy (9.649 kcal/mol) and the highest total hydrogen bond energy (62.83 kJ/mol), which were related to the positional isomeric effect on the hypoglycemic effect in mice. This work introduced a new means to develop antihyperglycemic agents in the field of multivalent glycomimetics.