Considerable interest has been attracted in oleanolic acid and its analogues because of their hypoglycemic activity. In this study, a series of novel oleanolic acid analogues against α-glucosidase were synthesized and their biological activities were evaluated in vitro and in vivo. In vitro α-glucosidase inhibition activity results indicated that most of the designed analogues exhibited prominent inhibition activities, especially compounds 10, 15, 16 and 26 which with the IC50 values of 0.33 ± 0.01, 0.98 ± 0.06, 0.69 ± 0.01 and 0.72 ± 0.21 μM, respectively. Enzyme kinetic studies on the most potent compounds reveled that derivatives 10, 15, 16 and 26 were noncompetitive inhibitors. Moreover, the docking studies were carried out to prove that the four compounds could interact with the hydrophobic region of the active pocket and form hydrogen bonds to enhance the binding affinity of them with the α-glucosidase. Cytotoxicity evaluation assay demonstrated a high level of safety profile of the active compounds (10, 15, 16 and 26) against normal 3T3 cell line. Furthermore, the in vivo actual pharmacological potential studies on derivatives 10, 15, 16 and 26 showed that the hypoglycemic effects of them were comparable to that of positive control, acarbose.