In order to promote the development and utilization of the products related to the ethanolic extract of Tricholoma matsutake (T. matsutake), aqueous ethanolic solutions with different volume fractions of ethanol were used to extract T. matsutake. The contents of polysaccharides, polyphenols and total flavonoids were measured, and the scavenging abilities of T. matsutake ethanolic extracts towards DPPH, hydroxyl, and ABTS+ radicals and the inhibitory effect against α-glucosidase were determined. The inhibition types and mechanisms of the extracts were examined by enzyme kinetic analysis, UV spectroscopy, fluorescence spectroscopy and infrared spectroscopy. The results showed that the content of the polysaccharides in the ethanolic extract obtained with 70% (V/V) ethanol was the highest (46.45%). The contents of polyphenols and total flavonoids in the water extract were the highest (13.92% and 1.03%, respectively). The scavenging abilities of the T. matsutake ethanolic extracts for DPPH, hydroxyl and ABTS free radicals showed a dose-dependent manner, with the IC50 values being 1.03, 0.49 and 0.87 mg/mL, respectively. The ethanolic extract obtained with 70% (V/V) ethanol exhibited the greatest inhibitory effect against α-glucosidase, with the inhibition rate being 28.93% at an extract mass concentration of 0.2 mg/mL. The ethanolic extracts obtained from the study all had good antioxidant and α-glucosidase inhibitory activities, and inhibited enzyme activity in the form of anti-competitive mixed-type enzyme inhibition, thereby affecting the microenvironment of the enzyme and changing its secondary structure. The ethanolic extracts can be used for the development of natural antioxidants and hypoglycemic agents. This research provides a reference for in-depth studies on T. matsutake ethanol extracts.