Morchella esculenta protein hydrolysate (MPH) from a valued medicinal and edible fungus M. esculenta (L.) is an excellent material for functional food development. To promote MPH utilization, selenization of MPH was performed by applying a simple and environmentally friendly microwave irradiation procedure. The physicochemical characteristics of selenized MPH (Se-MPH) were investigated by SEM-EDX, FTIR, CD, and amino acid analyzer, and its biological activity were assessed by ABTS, DPPH, H2O2 scavenging, and reducing power assays, as well as alpha-glucosidase, alpha-amylase, and tyrosinase inhibition tests. The results showed that MPH was successfully selenized, Se content in Se-MPH reached 59.0 +/- 0.64 mg/g, and amino groups, hydroxyl groups, and sulfur atoms of methionine residues in the MPH molecule may participate in selenization. Furthermore, Se-MPH exhibited significantly enhanced antioxidant, antidiabetic, and tyrosinase inhibitory activities, compared with the native MPH and microwave-irradiated MPH. Thus, the microwave-assisted selenization is a feasible strategy for preparing organic Se and improving the biological activity of MPH.