Amomum tsao-ko Crevost et Lemarie (A. tsao-ko) is a well-known dietary spice and traditional Chinese medicine. This study aimed to identify the flavonoids in A. tsao-ko and evaluate their antioxidant and antidiabetic activities in in vitro and in vivo studies. A. tsao-ko methanol extracts possessed a high flavonoid content (1.21 mg QE per g DW) and a total of 29 flavonoids were identified by employing UPLC-MS/MS. In vitro, A. tsao-ko demonstrated antioxidant activity (ORAC value of 34276.57 μM TE/100 g DW, IC50 of ABTS of 3.49 mg mL-1 and FRAP value of 207.42 μM Fe2+ per g DW) and α-amylase and α-glucosidase inhibitory ability with IC50 values of 14.23 and 1.76 mg mL-1, respectively. In vivo, type 2 diabetes mellitus (T2DM) models were induced by a combined high-fat diet (HFD) and streptozotocin (STZ) injection in rats. Treatment with the A. tsao-ko extract (100 mg freeze-dried powder per kg bw) for 6 weeks could significantly improve impaired glucose tolerance, decrease the levels of fasting blood glucose (FBG), insulin, and malondialdehyde (MDA), and increase the superoxide dismutase (SOD) level. Histopathology revealed that the A. tsao-ko extract preserved the architecture and function of the pancreas. In conclusion, the flavonoid composition of A. tsao-ko exhibits excellent antioxidant and antidiabetic activity in vitro and in vivo. A. tsao-ko could be a novel natural material and developed as a related functional food and medicine in T2DM management.