Abstract: Objective: To investigate the inhibitory effect of Tremella fuciformis polysaccharide (TP) on pancreatic α-amylase and α-glucosidase and its action mechanism. Methods: TP with a total sugar content of 92.45% was obtained by alkaline extraction from dried Tremella fuciformis, enzymatic deproteinization and column chromatography separation in turn. The inhibitory effect of TP on pancreatic α-amylase and α-glucosidase was measured by visible spectrophotometry, and its action on the structures of these two enzymes was characterized by fluorescence spectrometry and circular dichroism. Results: TP could inhibit the activities of these two enzymes, and its inhibition on pancreatic α-amylase was significantly higher than that on α-glucosidase, and its half inhibitory concentrations (IC₅₀) on these two enzymes were 7.6835 and 16.9306 mg/mL, respectively. TP inhibited the activity of the enzymes by interacting with them. TP interacted strongly with pancreatic α-amylase. It could statically quench pancreatic α-amylase and change its secondary structure. However, TP interacted weakly with α-glucosidase and could not change its secondary structure. Conclusion: TP inhibited the activity of starch-digestive enzymes by interacting with them.