A chemical derivative of trypsin, selenotrypsin, was prepared to imitate glutathione peroxidase (GPX) by converting active serine residues in the active site of trypsin into selenocysteines. The strategy for preparation of selenotrypsin contained selective sulfonation by phenylmethanesulfonyl fluoride and nucleophilic substitution by NaHSe. Selenotrypsin displayed enzyme activity of GPX, which is 150U/(mol. The kinetic properties of selenotrypsin was demonstrated to be similar to that of native GPX.