Abstract: Objectives: This study aimed to optimize the selenization process of polysaccharides derived from Dendrobium huoshanense (DHP) and to investigate the inhibitory effects and mechanisms of both DHP and selenium-enriched DHP (Se-DHP) on α-amylase activity. Methods: To investigate the impact of factors such as the mass ratio of sodium selenite to DHP, reaction time, nitric acid concentration, and reaction temperature on the selenium content in Se-DHP, a single factor experiment was conducted. Optimization of the Se-DHP preparation conditions was achieved using response surface methodology, with selenium content as the response variable. Additionally, the inhibitory effects of DHP and Se-DHP on α-amylase activity were compared, and their inhibition mechanisms were examined through fluorescence quenching experiments. Results: The optimal conditions for selenization were identified as follows: reaction temperature of 70 ℃, reaction time of 460 minutes, a mass ratio of sodium selenite to DHP of 1:1, and a nitric acid concentration of 0.5%. Under these conditions, the selenium content in Se-DHP was 9223.51 μg/g, closely matching the predicted value of 9280.59 μg/g. Compared to DHP, Se-DHP demonstrated a significantly enhanced inhibitory effect on α-amylase. Fluorescence spectroscopy revealed that both DHP and Se-DHP exhibited static quenching mode for α-amylase. Conclusions: The findings provide a theoretical framework for the preparation of Se-DHP with high-selenium content and offer a reference for the potential use of selenium polysaccharides from DHP as a new selenium supplement in medicinal and nutritional applications.