Multivalent glycosidase inhibitors based on 1-deoxynojirimycin derivatives against alpha-glucosidases have been rapidly developed. Nonetheless, the mechanism based on self-assembled multivalent glucosidase inhibitors in living systems needs to be further studied. It remains to be determined whether the self-assembly possesses sufficient stability to endure transit through the small intestine and subsequently bind to the glycosidases located therein. In this paper, two amphiphilic compounds, 1-deoxynojirimycin and alpha-peptoid conjugates (LP-4DNJ-3C and LP-4DNJ-6C), were designed. Their self-assembling behaviors, multivalent alpha-glucosidase inhibition effect, and fluorescence imaging on living organs were studied. LP-4DNJ-6C exhibited better multivalent alpha-glucosidase inhibition activities in vitro. Moreover, the self-assembly of LP-4DNJ-6C could effectively form a complex with Nile red. The complex showed fluorescence quenching effect upon binding with alpha-glucosidases and exhibited potent fluorescence imaging in the small intestine. This result suggests that a multivalent hypoglycemic effect achieved through self-assembly in the intestine is a viable approach, enabling the rational design of multivalent hypoglycemic drugs.