Selenoprotein plays a crucial role in immune cells and inflammatory regulation. However, as a protein drug that is easily denatured or degraded in the acidic environment of the stomach, efficient oral delivery of selenoprotein is a great challenge. Herein, we innovated an oral hydrogel microbeads-based biochemical strategy that can in situ synthesize selenoproteins, therefore bypassing the necessity and harsh conditions for oral protein delivery while effectively generating selenoproteins for therapeutic applications. The hydrogel microbeads were synthesized by coating hyaluronic acid-modified selenium nanoparticles with a protective shell of calcium alginate (SA) hydrogel. We tested this strategy in mice with inflammatory bowel disease (IBD), one of the most representative diseases related to intestinal immunity and microbiota. Our results revealed that hydrogel microbeads-mediated in situ synthesis of selenoproteins could prominently reduce proinflammatory cytokines secretion and mediate immune cells (e.g., reduce neutrophils and monocytes and increase immune regulatory T cells) to effectively relieve colitis-associated symptoms. This strategy was also able to regulate gut microbiota composition (increase probiotics abundance and suppress detrimental communities) to maintain intestinal homeostasis. Considering intestinal immunity and microbiota widely associated with cancers, infections, inflammations, etc., this in situ selenoprotein synthesis strategy might also be possibly applied to broadly tackle various diseases.