Tea is consumed worldwide and can reduce the risk of diabetes whereas the hypoglycemic compounds in tea remain unclear. Herein, the chemical compositions comparison and inhibition assays indicated that the inhibitory effect of tea against α-amylase was positively related with the enzyme-mediated oxidation degree of tea and black teas containing high contents of theaflavins exhibited the strongest in vitro inhibitory effect. Affinity selection-mass spectrometry-based high-throughput screening revealed that four theaflavins specifically bind to α-amylase. Compared to other compounds, theaflavins showed one order of magnitude higher in vitro inhibitory effects; thus, theaflavins are possibly the dominant α-amylase inhibitors in tea. A series of “α-amylase–theaflavins” interaction studies jointly demonstrated that theaflavins noncompetitively inhibit α-amylase activity by interacting with the amino acid residues of α-amylase and then inducing changes in the secondary structure. Furthermore, theaflavins (10 mg/kg/d) and black tea extracts (100 mg/kg/d) exhibited comparable hypoglycemic effects as acarbose in diabetic mice.